Single copy genomic hybridization probes and method of generating same

ABSTRACT

Nucleic acid (e.g., DNA) hybridization probes are described which comprise a labeled, single copy nucleic acid which hybridizes to a deduced single copy sequence interval in target nucleic acid of known sequence. The probes, which are essentially free of repetitive sequences, can be used in hybridization analyses without adding repetitive sequence-blocking nucleic acids. This allows rapid and accurate detection of chromosomal abnormalities. The probes are preferably designed by first determining the sequence of at least one single copy interval in a target nucleic acid sequence, and developing corresponding hybridization probes which hybridize to at least a part of the deduced single copy sequence. In practice, the sequences of the target and of known genomic repetitive sequence representatives are compared in order to deduce locations of the single copy sequence intervals. The single copy probes can be developed by any variety of methods, such as PCR amplification, restriction or exonuclease digestion of purified genomic fragments, or direct synthesis of DNA sequences. This is followed by labeling of the probes and hybridization to a target sequence.

RELATED APPLICATION

[0001] This is a continuation-in-part of Ser. No. 09/573,080 filed May16, 2000.

SEQUENCE LISTING

[0002] A Sequence Listing containing 613 sequences in the form of acomputer readable ASCII file in connection with the present invention isincorporated herein by reference and appended hereto as one (1) originalcompact disk in accordance with 37 CFR 1.821(c), an identical copythereof in accordance with 37 CFR 1.821(e), and one (1) identical copythereof in accordance with 37 CFR 1.52(e).

COMPUTER PROGRAM LISTING APPENDIX

[0003] A computer program listing appendix containing the source code ofa computer program that may be used with the present invention isincorporated herein by reference and appended hereto as one (1) originalcompact disk, and an identical copy thereof, containing a total of 3files as follows: Date of Creation Size (Bytes) File Name Apr. 18, 200126 KB FINDI.PL Apr. 18, 2001 19 KB PRIM.IN Apr. 18, 2001 20 KB PRIM.WKG

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] The present invention is broadly concerned with a method fordesigning single copy hybridization probes useful in the fields ofcytogenetics and molecular genetics for determining the presence ofspecific nucleic acid sequences in a sample of eukaryotic origin, e.g.,the probes may be used to analyze specific chromosomal locations by insitu hybridization as a detection of acquired or inherited geneticdiseases. More particularly, the invention pertains to such probes,hybridization methods of use thereof and techniques for developing theprobes, where the probes are essentially free of genomic repeatsequences, thereby eliminating the need for disabling of repetitivesequences which is required with conventional probes.

[0006] 2. Description of the Prior Art

[0007] Chromosome abnormalities are associated with various geneticdisorders, which may be inherited or acquired. These abnormalities areof three general types, extra or missing individual chromosomes(aneuploidy), extra or missing portions of chromosomes (includingdeletions, duplications, supernumerary and marker chromosomes), orchromosomal rearrangements. The latter category includes translocations(transfer of a piece from one chromosome onto another chromosome),inversions (reversal in polarity of a chromosomal segment), insertions(transfer of a piece from one chromosome into another chromosome) andisochromosomes (chromosome arms derived from identical chromosomalsegments). The abnormalities may be present only in a subset of cells(mosaicism), or in all cells. Inherited or constitutional abnormalitiesof various types occur with a frequency of about one in every 250 humanbirths, with results which may be essentially benign, serious or evenlethal. Chromosomal abnormalities are common and often diagnostic inacquired disorders such as leukemia and other cancers.

[0008] Hybridization probes have been developed in the past forchromosome analysis and diagnosis of abnormalities. The probes comprisecloned or amplified genomic sequences or cDNA. For example, U.S. Pat.Nos. 5,447,841, 5,663,319 and 5,756,696 describe hybridization probes inthe form of labeled nucleic acids which are complementary to nucleicacid segments within target chromosomal DNA. However, these probescontain repetitive sequences and therefore must be used in conjunctionwith blocking nucleic acids which are substantially complementary torepetitive sequences in the labeled probes. That is, these prior artprobes are either pre-reacted with the blocking nucleic acids so as tobind and block the repetitive sequences therein, or such blockingnucleic acids are present in the hybridization reaction mixture. If therepetitive sequences in the probes are not disabled in some manner, theprobes will react with the multiple locations in the target chromosomalDNA where the repetitive sequences reside and will not specificallyreact with the single copy target sequences. This problem isparticularly acute with interspersed repeat sequences which are widelyscattered throughout the genome, but also is present with tandem repeatsclustered or contiguous on the DNA molecule. The requirement for repeatsequence disabilization by using complementary blocking nucleic acidsreduces the sensitivity of the existing probes. Reliable, easilydetectable signals require DNA probes of from about 40-100 kb.

[0009] The prior art also teaches that cloned probes presumed to containsingle copy sequences can be identified based on their lack ofhybridization to radiolabeled total genomic DNA. In these other studies,hybridization is first performed with probes that contain pools ofclones in which each recombinant DNA clone has been individuallyselected so that it hybridizes to single-copy sequences or very low copyrepetitive sequences. A prerequisite step in this prior art is toidentify single copy sequences by experimental hybridization of labeledgenomic DNA to a candidate DNA probe by Southern or dot-blothybridization. Positive hybridization with labeled total genomic DNAusually indicates that the candidate DNA probe contains a repetitivesequence and eliminates it from consideration as a single copy probe.Furthermore, an experimental hybridization of a DNA probe with totalgenomic DNA may fail to reveal the presence of multicopy repetitivesequences that are not abundant (<100 copies) or are infrequent in thegenome. Such sequences represent a small fraction of the labeled genomicDNA and the signal they contribute will be below the limits ofdetection.

[0010] It has also been suggested to physically remove repeat sequencesfrom probes by experimental procedures (Craig et al., Hum. Genet.,100:472-476 (1997); Durm et al., Biotech., 24:820-825 (1998)). Thisprocedure involves prehybridizing a polymerase chain reaction(PCR)-amplified genomic probe with an excess of purified repetitivesequence DNA prior to applying the probe to the DNA target. Theresulting purified probe is depleted of repetitive sequences. Thisprocedure is in principle very similar to other procedures that disablethe hybridization of repetitive sequences in probes, but the techniqueis time-consuming and does not provide any advantages over the probesdescribed in Pat. Nos. 5,447,841 and 5,756,696.

SUMMARY OF THE INVENTION

[0011] The present invention overcomes the problem outlined above andprovides nucleic acid (e.g., DNA) hybridization probes comprising alabeled, single copy nucleic acid which hybridizes with a deduced singlecopy sequence interval in target nucleic acid of known sequence.Generally speaking, the probes of the invention are designed bycomparing the sequence of a target nucleic acid with known repeatsequences in the genome of which the target is a part; with thisinformation it is possible to deduce the single copy sequences withinthe target (i.e., those sequences which are essentially free of repeatsequences which, due to the lack of specificity, can mask thehybridization signal of the single copy sequences). As can beappreciated, these initial steps require knowledge of the sequences bothof the target and genomic repeats, information which is increasinglyavailable owing to the Human Genome Project and related bioinformaticstudies. Furthermore, readily available computer software is used toderive the necessary single copy sequences.

[0012] The probes hereof are most preferably complementary to the targetsequence, i.e., there is a 100% complementary match between the probenucleotides and the target sequence. More broadly, less than 100%correspondence probes can be used, so long as the probes adequatelyhybridize to the target sequence, i.e., there should be at least about80% sequence identity between the probe and a sequence which is acomplement to target sequences, more preferably at least about 90%sequence identity.

[0013] Nucleic acid fragments corresponding to the deduced single copysequences can be generated by a variety of methods, such as PCRamplification, restriction or exonuclease digestion of purified genomicfragments, or direct nucleic acid synthesis. The single copy fragmentsare then purified to remove any potentially contaminating repeatsequences, such as, for example, by electrophoresis or denaturing highpressure liquid chromatography; this is highly desirable because iteliminates spurious hybridization and detection of unrelated genomicsequences.

[0014] The probe fragments may then be cloned into a recombinant DNAvector or directly labeled. The probe is preferably labeled by nicktranslation using a modified or directly labeled nucleotide. The labeledprobe is then denatured and hybridized, preferably to fixed chromosomalpreparations on microscope slides or alternately to purified nucleicacid immobilized on a filter, slide, DNA chip, or other substrate. Theprobes can then be hybridized to chromosomes according to conventionalfluorescence in situ hybridization (FISH) methods such as thosedescribed in U.S. Pat. Nos. 5,985,549 or 5,447,841; alternately, theycan be hybridized to immobilized nucleic acids according to thetechniques described in Pat. Nos. 5,110,920 or 5,273,881. Probe signalsmay be visualized by any of a variety of methods, such as thoseemploying fluorescent, immunological or enzymatic detection reagents.

[0015] Use of the probes of the invention permits more precisechromosomal breakpoint determinations, to a level of resolutionheretofore unobtainable by in situ hybridization. In such analyses,initial probe sets can be prepared from regions believed to be onopposite sides of the breakpoint. After an initial assay to confirmthis, successive additional probes closer to the breakpoint can bedesigned, using the single copy strategy. In this fashion, the preciseregion of the breakpoint can be determined.

[0016] It has been found that use of putative single copy probes candetermine the existence of heretofore unknown repeat sequences in agenome. The heretofore unknown repeated sequence families can then beincluded in the repetitive sequence database so that these sequences canbe used in the design of subsequent single copy probes.

[0017] It was also found that probes may contain sequences that areduplicated or triplicated in the genome which can have strongerhybridization due to the increased length of the target sequence. Also,these duplicons or triplicons can be confirmed, as such, using singlecopy probes which is more difficult with available commercial probes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1-12 are respective CCD camera images of FISH experimentswherein various gene-specific digoxigenin-dUTP labeled probes werehybridized on metaphase cells and detected with rhodamine conjugatedantibody to digoxigenin and where the chromosomes were counterstainedwith 4,6-diamidino-2-phenylindole (DAPI). Chromosomes with one or bothchromatids hybridized are indicated by arrows whereas a star indicatesthe absence of normally expected hybridizations. In particular,

[0019]FIG. 1 illustrates hybridization results using the 5170 bp HIRAprobe described in Example 1, and wherein the probe was reacted withpurified repetitive DNA sequences;

[0020]FIG. 2 illustrates a comparative hybridization identical to thatdepicted in FIG. 1, using the same 5170 bp HIRA probe but withoutpre-reaction with purified repetitive DNA sequences;

[0021]FIG. 3 illustrates hybridization results using the 3544 bp15q11-q13 probe pre-reacted with purified repetitive DNA;

[0022]FIG. 4 illustrates results in a comparative experiment using the3544 bp 15q11-q13 probe without pre-reaction with purified repetitiveDNA;

[0023]FIG. 5 illustrates hybridization results using the 4166 bp, 3544bp and 2290 bp 15q11-q13 probes described in Example 2, withoutpre-reaction with purified repetitive DNA sequences;

[0024]FIG. 6 illustrates hybridization results using the 5170 bp, 3691bp, 3344 bp and 2848 bp HIRA probes described in Example 1 withoutpre-reaction with purified repetitive DNA sequences;

[0025]FIG. 7 illustrates hybridization results using the 4823 bp 1p36.3probe described in Example 2 on metaphase cells of a normal individual,with pre-reaction with purified repetitive DNA sequences;

[0026]FIG. 8 illustrates a comparative hybridization result using the4823 bp 1p36.3 probe of FIG. 7 without pre-reaction with purifiedrepetitive DNA sequences;

[0027]FIG. 9 illustrates hybridization results using the 4724 bp and4823 bp 1p36.3 probes described in Example 2 with pre-reaction withpurified repetitive DNA sequences, and wherein single copyhybridizations were observed on homologous pairs of chromosome 1s;

[0028]FIG. 10 illustrates a comparative hybridization result using the4724 bp and 4823 bp 1p36.3 probes described in Example 2 withoutpre-reaction with purified repetitive DNA sequences, and depicting thesame single copy hybridizations shown in FIG. 9;

[0029]FIG. 11 illustrates hybridization results using the 4166 bp, 3544bp and 2290 bp 15q11-q13 probes described in Example 2 withoutpre-reaction with purified DNA sequences on metaphase cells of a patientaffected with Prader-Willi syndrome and known to harbor a deletion of15q11-q13 sequences for one chromosomal allele, with a star indicatinglack of hybridization at the deleted chromosome position and with thearrow indicating hybridization to a single chromosome;

[0030]FIG. 12 illustrates hybridization results using the 3691 bp, 3344bp and 2848 bp HIRA probes described in Example 1 without pre-reactionwith purified DNA sequences on metaphase cells of a patient affectedwith DiGeorge/Velo-Cardio-Facial Syndrome (VCFS) known to harbor adeletion of 22q11.2 sequences, wherein the star indicating lack ofhybridization at the deleted chromosome position and the arrowindicating a normal homolog;

[0031]FIG. 13 is a scatterplot of base pair coordinates versus singlecopy probe lengths found in the Breakage Cluster Region gene (BCR)promoter found on chromosome 22, the disruption of which is common incases of chronic adult myeloid leukemia and in some cases of acutelymphoblastic leukemia, as described in Example 4;

[0032]FIG. 14 is a scatterplot of base pair coordinates versus singlecopy probe lengths found in the ABL1 gene on chromosome 9, thedisruption of which is common in cases of chronic adult myeloid leukemiaand in some cases of acute lymphoblastic leukemia, as described inExample 4;

[0033]FIG. 15 is a CCD camera image of a FISH experiment usingchromosome 9q34-specific, digoxigenin-dUTP labeled probes from the ABL1oncogene (SEQ ID Nos. 520-525), and detected with rhodamine (red)conjugated antibody to digoxigenin with DAPI stained metaphase cellsfrom a patient with chronic myelogenous leukemia (CML), illustrating theuse of probes downstream of the site of fusion between ABL1 gene and BCRgene used to make a precise chromosomal breakpoint determination asexplained in Example 4 wherein the derivative chromosome 22 and normalchromosome 9 are indicated; and

[0034]FIG. 16 is a CCD camera image of a FISH experiment usingchromosome 9q34-specific, digoxigenin-dUTP labeled probes from the ABL1oncogene (SEQ ID Nos. 516-525) and detected with rhodamine (red)conjugated antibody to digoxigenin, with DAPI stained metaphase cellsfrom a patient with CML, illustrating the use of probes from each sideof the site of fusion between BCR and ABL1 genes used to make a precisechromosomal 9q34 breakpoint determination as explained in Example 4,wherein the derivative chromosome 22, derivative chromosome 9 and normalchromosome 9 are indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0035] The present invention is concerned with nucleic acid (e.g., DNA)hybridization probes useful for detection of genetic or neoplasticdisorders. The probes are in the form of labeled nucleic acid fragmentsor a collection of labeled nucleic acid fragments whose hybridization toa target sequence can be detected. The invention also pertains tomethods of developing, generating and labeling such probes, and to usesthereof.

[0036] The labeled probes hereof may be used with any nucleic acidtarget that may potentially contain repetitive sequences. These targetsequences may include, but are not limited to chromosomal or purifiednuclear DNA, heteronuclear RNA, or mRNA species that contain repetitivesequences as integral components of the transcript. In the ensuingdetailed explanation, the usual case of a DNA target sequence and DNAprobes is discussed; however, those skilled in the art will understandthat the discussion is equally applicable (with art-recognizeddifferences owing to the nature of the target sequences and probes) toother nucleic acid species.

[0037] An important characteristic of the probes of the invention isthat they are composed of “single copy” or “unique” DNA sequences whichare both complementary to at least a portion of the target DNA region ofinterest and are essentially free of sequences complementary to repeatsequences within the genome of which the target region is a part.Accordingly, a probe made up of a single copy or unique sequence iscomplementary to essentially only one sequence in the correspondinggenome.

[0038] Very recently, it has been discovered that the human genomecontains highly similar domains which have been termed duplicons whenthey are present in two non-allelic copies or triplicons when present inthree copies in the genome (Ji et al., Genome Res., 10:597-610 (2000)).Duplication or triplication of chromosomal domains containing suchsequences were recent evolutionary events, based on the fact thatnon-human primates, in some instances, do not contain multiple copies ofthese sequences, and on the high degree of sequence similarity betweendifferent copies of paralogous sequences. These low copy duplicons (ortriplicons) are to be distinguished from classic repetitive sequencefamilies, which tend to either be interspersed throughout the genome orto be tandemly reiterated hundreds to thousands of times in the samechromosomal interval; therefore, probes from duplicons or triplicons arefor purposes of the present invention deemed to be within the ambit of“single copy” probes.

[0039] These duplicons or triplicons have evolved so recently that thesequence and organization of an entire genomic domain—which comprisescomplex, near-single copy segments and adjacent members of knownrepetitive sequence families—is completely conserved in each duplicon ortriplicon segment. Duplicon and triplicon lengths of several kilobasesto megabase sizes have been reported (International Genome SequencingConsortium, Nature, 409:860-922 (2001)). The duplicons/triplicons areoften tandemly arranged, and are almost always present on the samechromosome, and are therefore clustered in the genome. The length of theinterval separating paralogous probe sequences is dictated by the sizeof the duplicated/triplicated domain, the orientation of the duplicons(or triplicons) relative to each other (ie. direct or inverted), and thelength of unrelated sequence intervals, if any, which separate theduplicons/triplicons.

[0040] In the context of the present invention, the term “single copy”with reference to a nucleic acid sequence refers to a sequence which isstrictly unique (i.e., which is complementary to one and one onlysequence in the corresponding genome) but also covers duplicons andtriplicons. Stated otherwise, a “single copy” probe in preferred formswill hybridize to three or less locations in the genome.

[0041] As used herein, a “repeat sequence” is a sequence whichrepeatedly appears in the genome of which the target DNA is a part, witha sequence identity between repeats of at least about 60%, morepreferably at least about 80%, and which is of sufficient length or hasother qualities which would cause it to interfere with the desiredspecific hybridization of the probe to the target DNA (i.e., the probewould hybridize with multiple copies of the repeat sequence). Generallyspeaking, a repeat sequence appears at least about 10 times in thegenome (more preferably at least about 50 times, and most preferably atleast about 200 times) and has a length of at least about 50nucleotides, and more preferably at least about 100 nucleotides. Repeatsequences can be of any variety (e.g., tandem, interspersed, palindromicor shared repetitive sequences with some copies in the target region andsome elsewhere in the genome), and can appear near the centromeres ofchromosomes, distributed over a single chromosome, or throughout some orall chromosomes. Normally, with but few exceptions, repeat sequences donot express physiologically useful proteins.

[0042] Repetitive sequences occur in multiple copies in the haploidgenome. The number of copies can range from at least about 10 tohundreds of thousands, wherein the Alu family of repetitive DNA areexemplary of the latter numerous variety. The copies of a repeat may beclustered or interspersed throughout the genome. Repeats may beclustered in one or more locations in the genome, for example,repetitive sequences occurring near the centromeres of each chromosome,and variable number tandem repeats (VNTRS) (Nakamura et al., Science,235:1616 (1987)); or the repeats maybe distributed over a singlechromosome for example, repeats found only on the X chromosome asdescribed by Bardoni et al. (Cytogenet. Cell Genet., 46:575 (1987)); orthe repeats may be distributed over all the chromosomes, for example,the Alu family of repetitive sequences.

[0043] Simple repeats of low complexity can be found within genes butare more commonly found in non-coding genomic sequences. Such repeatedelements consist of mono-, di-, tri-, tetra-, or penta-nucleotide coresequence elements arrayed in tandem units. Often the number of tandemunits comprising these repeated sequences varies at the identicallocations among genomes from different individuals. These repetitiveelements can be found by searching for consecutive runs of the coresequence elements in genomic sequences.

[0044] As used herein, “sequence identity” refers to a relationshipbetween two or more polynucleotide sequences, namely a referencesequence and a given sequence to be compared with the referencesequence. Sequence identity is determined by comparing the givensequence to the reference sequence after the sequences have beenoptimally aligned to produce the highest degree of sequence similarity,as determined by the match between strings of such sequences. Upon suchalignment, sequence identity is ascertained on a position-by-positionbasis, e.g., the sequences are “identical” at a particular position ifat that position, the nucleotides are identical. The total number ofsuch position identities is then divided by the total number ofnucleotides or residues in the reference sequence to give % sequenceidentity. Sequence identity can be readily calculated by known methods,including but not limited to, those described in Computational MolecularBiology, Lesk A. N., ed., Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects, Smith D. W., ed.,Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I, Griffin A. M., and Griffin H. G., eds., Humana Press, New Jersey(1994); Sequence Analysis in Molecular Biology, von Heinge G., AcademicPress (1987); Sequence Analysis Primer, Gribskov M. and Devereux J.,eds., M. Stockton Press, New York (1991); and Carillo H., and Lipman D.,SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determinethe sequence identity are designed to give the largest match between thesequences tested. Methods to determine sequence identity are codified inpublicly available computer programs which determine sequence identitybetween given sequences. Examples of such programs include, but are notlimited to, the GCG program package (Devereux et al., Nuc. Ac. Res.,12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul et al., J. Molec.Biol., 215:403-410 (1990)). The BLASTX program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul et al., NCBI, NLM,NIH, Bethesda, Md. 20894; Altschul et al., J. Molec. Biol., 215:403-410(1990)). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 95% “sequence identity” to a reference nucleotide sequence, itis intended that the nucleotide sequence of the given polynucleotide isidentical to the reference sequence except that the given polynucleotidesequence may include up to 5 differences per each 100 nucleotides of thereference nucleotide sequence. In other words, in a polynucleotidehaving a nucleotide sequence having at least 95% identity relative tothe reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.Inversions in either sequence are detected by these computer programsbased on the similarity of the reference sequence to the antisensestrand of the homologous test sequence. These variants of the referencesequence may occur at the 5′ or 3′ terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

[0045] The single copy probes of the invention preferably should have alength of at least about 50 nucleotides, and more preferably at leastabout 100 nucleotides. Probes of this length are sufficient for Southernblot analyses. However, if other analyses such as FISH are employed, theprobes should be somewhat longer, i.e., at least about 500 nucleotides,and more preferably at least about 2000 nucleotides in length. Theprobes can be used to detect virtually any type of chromosomalrearrangement, such as deletions, duplications, insertions, additions,inversions or translocations.

[0046] In order to develop probes in accordance with the invention, thesequence of the target DNA region must be known. The target region maybe an entire chromosome or only portions thereof where rearrangementshave been identified. With this sequence knowledge, the objective is todetermine the boundaries of single copy or unique sequences within thetarget region. This is preferably accomplished by inference from thelocations of repetitive sequences within the target region. Normally,the sequence of the target region is compared with known repeatsequences from the corresponding genome, using available computersoftware. Once the repeat sequences within the target region areidentified, the intervening sequences are deduced to be single copy(i.e., the sequences between adjacent repeat sequences).

[0047] Optimal alignment of the target and repetitive sequences forcomparison may be conducted by the local homology algorithm of Smith etal., Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithmof Needleman et al., J. Mol. Biol., 48:443 (1970). The results obtainedfrom the heuristic methods (Pearson et al., Proc. Natl. Acad. Sci.,85:244 (1988); Altschul et al., J. Molec. Biol., 215:403-410 (1990)) aregenerally not as comprehensive as the methods of Smith et al. (1981) andNeedleman et al., (1970). However, they are faster than these methods.

[0048] Once the single copy sequence information is obtained, certain ofthe single copy sequences (normally the longest) are used to designhybridization probes. In this regard, probes may be of varying“complexity” as defined by Britten et al., Methods of Enzymol., 29:363(1974) and as further explained by Cantor et al., Biophysical Chemistry:Part III: The Behavior of Biological Macromolecules, pp. 1228-1230. Thecomplexity of selected probes is dependent upon the application forwhich it is designed. In general, the larger the target area, the morecomplex the probe. The complexity of a probe needed to detect a set ofsequences will decrease as hybridization sensitivity increases. At highsensitivity and low background, smaller and less complex probes can beused.

[0049] With current hybridization techniques, it is possible to obtainreliable, easily detectable signals with relatively small probes inaccordance with the invention. A readily detectable signal was obtainedwith a probe on the order of 2 kb in length, using FISH technology. Thissensitivity of the present method is improved compared to the prior art(U.S. Pat. No. 5,756,696) because the probes of the present inventionare homogeneous single copy sequences. However, smaller amplifiedsegments, each comprising non-repetitive sequences, may also be used incombination as probes to achieve adequate signals for in situhybridization. Complex single copy probes that hybridize to duplicatedor triplicated targets can also increase hybridization signals.

[0050] One application of the use of multiple fragment probes is in thedetection of translocations between different chromosomes.Proportionately increasing the complexity of the probe also permitsanalysis of multiple compact regions of the genome simultaneously. For asingle chromosome, the portion of the probe targeted to one side of thebreakpoint can be labeled and detected differently from that targeted tothe other side of the breakpoint so that the derivative or translocatedchromosome is detected by one label and is distinguishable from theintact normal chromosome which has both labels.

[0051] The invention makes it possible to produce single copy probes ata higher genomic density than possible using conventional probes.Chromosomes 21 and 22 have been comprehensively sequenced, and it hasbeen determined that adjacent single copy intervals tend to be clusteredon these chromosomes. For example, on chromosome 22,39% of single copyintervals are separated by only 500-1000 bp. Single copy intervals ≧2.3kb are separated, on average, by 29.2 kb on chromosome 21 and by 22.3 kbon chromosome 22.

[0052] In order to estimate the size of genomic intervals required todevelop single copy probes, the probability of detecting at least onesingle copy sequence in overlapping, uniform-length genomic intervals onchromosomes 21q and 22q was determined. Single copy segments ≧2.0 kb inlength are found in the majority of 100 kb genomic intervals of thesechromosomes (96% of chromosome 22q and 88% of chromosome 21q).Increasing the size of the genomic sequence to 150 kb results in 99%coverage of chromosome 22q and 96% of chromosome 21q. Therefore, singlecopy probes should be more or less 2 kb to ensure comprehensive coverage(at least once per 100-150 kb) of chromosomes 21 and 22. Assuming thatsingle copy sequences are similarly distributed on other chromosomes, itshould be feasible to develop probes for in situ hybridization analysisof most clinically relevant chromosomal rearrangements.

[0053] Once appropriate single copy sequences in the chromosomal regionof interest have been identified, PCR is preferably used for amplifyingthe appropriate DNA to obtain probes. PCR is a well known technique foramplifying specific DNA segments in geometric progression and reliesupon repeated cycles of DNA polymerase-catalyzed extension from a pairof oligonucleotide primers with homology to the 5′ end and to thecomplement of the 3′ end of the DNA segment to be amplified.

[0054] The nucleic acid (e.g., DNA) that serves as the PCR template maybe single stranded or double stranded, but when the DNA is singlestranded, it will typically be converted to double stranded. The lengthof the template DNA may be as short as 50 bp, but usually will be atleast about 100 bp long, and more usually at least about 150 bp long,and may be as long as 10,000 bp or longer, but will usually not exceed50,000 bp in length, and more usually will not exceed 20,000 bp inlength. The DNA may be free in solution, flanked at one or both endswith non-template DNA, present in a cloning vector such as a plasmid andthe like, with the only criteria being that the DNA be available forparticipation in the primer extension reaction. The template DNA may bederived from a variety of different sources, so long as it iscomplementary to the target chromosomal or immobilized DNA sequence. Theamount of template DNA that is combined with the other reagents willrange from about 1 molecule to 1 pmol, usually from about 50 moleculesto 0.1 pmol, and more usually from about 0.01 pmol to 100 fmol. Theoligonucleotide primers with which the template nucleic acid iscontacted will be of sufficient length to provide for hybridization tocomplementary template DNA under annealing conditions but will be ofinsufficient length to form stable hybrids with template DNA underpolymerization conditions. The primers will generally be at least about10 nucleotides (nt) in length, usually at least 15 nt in length and moreusually at least 16 nt in length and may be as long as 30 nt in lengthor longer, where the length of the primers will generally range from 18to 50 nt in length, usually from about 20 to 3 5 nt in length. The yieldof longer amplification products can be enhanced using primers of 30 to35 nt and high fidelity polymerases (described in U.S. Pat. No.5,436,149).

[0055] To maximize the signal intensity obtained during in situhybridization, primer sequence pairs are preferred which, uponamplification, produce a DNA fragment that spans nearly the entirelength of each single-copy genomic sequence interval. Hence, contiguousor closely spaced (software excludes pairs that are separated by ≦70% ofthe length of the single copy interval) primer pairs are generallyexcluded from consideration for producing probes for in situhybridization. With the exception of cytogenetic preparations, thiscriterion is generally not applicable for probes that are hybridized toimmobilized cloned or synthetic nucleic acid targets, since signalintensities of shorter probes are usually adequate due to the increasednumber of target molecules.

[0056] However, in order to optimize the yield and kinetics of the PCRreaction, the desired primer sequences are also subject to othercriteria. First, a primer sequence should not be substantiallyself-complementary or complementary to the second primer. In particular,potential primer sequences are excluded which could result in theformation of stable hybrids involving the 3′ terminus of the primer andeither another sequence in the same or the second primer (defined as ≧6base pairs). Additionally, the T_(m) of one member of the primer pairshould occur within 2° C. of its counterpart, which enables them todenature and anneal to the template nearly simultaneously. Software iswell known in the art to identify primer sequences that satisfy all ofthe preferred criteria (see for example:http://wwwgenome.wi.mit.edu/ftp/pub/software/primer.0.5/ orhttp://www.oligo.net/Oligo 6 tour.htm).

[0057] The PCR reaction mixture will normally further comprise anaqueous buffer medium which includes a source of monovalent ions, asource of divalent cations and a buffering agent. Any convenient sourceof monovalent ions, such as KCl, K-acetate, NH₄-acetate, K-glutamate,NH₄Cl, ammonium sulfate, and the like may be employed, where the amountof monovalent ion source present in the buffer will typically be presentin an amount sufficient to provide for a conductivity in a range fromabout 500 to 20,000, usually from about 1000 to 10,000, and more usuallyfrom about 3,000 to 6,000 microohms. The divalent cation may bemagnesium, manganese, zinc and the like, where the cation will typicallybe magnesium. Any convenient source of magnesium cation may be employed,including MgCl₂, Mg-acetate, and the like. The amount of Mg⁺²-present inthe buffer may range from 0.5 to 10 mM, but will preferably range fromabout 2 to 4 mM, more preferably from about 2.25 to 2.75 mM and willideally be at about 2.45 mM. Representative buffering agents or saltsthat may be present in the buffer include Tris, Tricine, HEPES, MOPS andthe like, where the amount of buffering agent will typically range fromabout 5 to 150 mM, usually from about 10 to 100 mM, and more usuallyfrom about 20 to 50 mM, where in certain preferred embodiments thebuffering agent will be present in an amount sufficient to provide a pHranging from about 6.0 to 9.5, where most preferred is pH 7.3 at 72° C.Other agents which may be present in the buffer medium include chelatingagents, such as EDTA, EGTA and the like.

[0058] Also present in the PCR reaction mixtures is a melting pointreducing agent, i.e., a reagent that lowers the melting point of DNA.Suitable melting point reducing agents are those agents that interferewith the hydrogen bonding interaction of two nucleotides, whererepresentative base pair destabilization agents include: betaine,formamide, urea, thiourea, acetamide, methylurea, glycinamide, and thelike, where betaine is a preferred agent. The melting point reducingagent will typically be present in amounts ranging from about 20 to 500mM, usually from about 50 to 200 mM and more usually from about 80 to150 mM.

[0059] In preparing the PCR reaction mixture, the various constituentcomponents may be combined in any convenient order. For example, thebuffer may be combined with primer, polymerase and then template DNA, orall of the various constituent components may be combined at the sametime to produce the reaction mixture.

[0060] Following preparation of the PCR reaction mixture, it issubjected to a plurality of reaction cycles, where each reaction cyclecomprises: (1) a denaturation step, (2) an annealing step, and (3) apolymerization step. The number of reaction cycles will vary dependingon the application being performed, but will usually be at least 15,more usually at least 20 and may be as high as 60 or higher, where thenumber of different cycles will typically range from about 20 to 40. Formethods where more than about 25, usually more than about 30 cycles areperformed, it may be convenient or desirable to introduce additionalpolymerase into the reaction mixture such that conditions suitable forenzymatic primer extension are maintained.

[0061] The denaturation step comprises heating the reaction mixture toan elevated temperature and maintaining the mixture at the elevatedtemperature for a period of time sufficient for any double stranded orhybridized nucleic acid present in the reaction mixture to dissociate.For denaturation, the temperature of the reaction mixture will usuallybe raised to, and maintained at, a temperature ranging from about 85 to100° C. usually from about 90 to 98° C., and more usually from about 93to 96° C. for a period of time ranging from about 3 to 120 seconds,usually from about 5 to 30 seconds.

[0062] Following denaturation, the PCR reaction mixture will besubjected to conditions sufficient for primer annealing to template DNApresent in the mixture. The temperature to which the reaction mixture islowered to achieve these conditions will usually be chosen to provideoptimal efficiency and specificity, and will generally range from about50 to 75° C., usually from about 55 to 70° C. and more usually fromabout 60 to 68° C. Annealing conditions will be maintained for a periodof time ranging from about 15 seconds to 30 minutes, usually from about30 seconds to 5 minutes.

[0063] Following annealing of primer to template DNA or during annealingof primer to template DNA, the reaction mixture will be subjected toconditions sufficient to provide for polymerization of nucleotides tothe primer ends in manner such that the primer is extended in a 5′ to 3′direction using the DNA to which it is hybridized as a template, i.e.conditions sufficient for enzymatic production of primer extensionproduct. To achieve polymerization conditions, the temperature of thereaction mixture will typically be raised to or maintained at atemperature ranging from about 65 to 75° C., usually from about 67 to73° C. and maintained for a period of time ranging from about 15 secondsto 20 minutes, usually from about 30 seconds to 5 minutes.

[0064] The above cycles of denaturation, annealing and polymerizationmay be performed using an automated device, typically known as a thermalcycler. Thermal cyclers that may be employed are described in U.S. Pat.Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610.

[0065] Based on all the previous criteria, a series of primers wereproduced and validated by PCR using genomic DNA from normal individuals.Knowledge of suitable primers will necessarily define the correspondingPCR-produced probes in accordance with the invention. Thus, adjacentpairs of sequences identified as SEQ ID Nos. 429-446 and 480-613,beginning with SEQ ID No.429, are respective forward/reverse PCR primersdeveloped for the production of specific useful probes. Hence, a usefulprobe may be produced using a combination of SEQ ID Nos. 429 and 430,and additional probes are defined by the succeeding pairs of adjacentSEQ IDs. Broadly speaking, certain preferred probes of the inventionshould have at least about 80% sequence identity, and more preferablyabout 90% sequence identity, relative to the probes defined by theabove-described adjacent pairs of primer sequences.

[0066] In addition to the PCR, DNA fragments corresponding to uniquesequences can also be obtained by a variety of other methods, includingbut not limited to deletion mutagenesis, restriction digestion, directsynthesis and DNA ligation.

[0067] If the genomic fragment is obtained by amplification orpurification from DNA containing repetitive sequences, the fragment mustthen be purified prior to labeling and hybridization. Purification ofhomogeneously-sized DNA fragments can be accomplished by a variety ofmethods, including but not limited to electrophoresis and high pressureliquid chromatography. In the preferred method, amplified fragments areseparated according to size by gel electrophoreses in Seakem LE Agaroseusing Tris Acetate buffer (Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual [Cold Spring Harbor Laboratory Press,1989]), stained with a dye such as ethidium bromide or Syber-Green,visualized with ultraviolet light (300 nm), excised from the gel using ascalpel. Each DNA fragment is then recovered from the gel fragment usinga Micro-con 100 (Millipore, Watertown, Mass.) column by spincentrifugation.

[0068] Phenol-chloroform extraction of the amplified DNA is not anadequate method of purification. When this approach was tested, thispurification technique resulted in nonspecific hybridization to allchromosomes along their entire length, which is consistent with thepattern produced by hybridization of repetitive sequences (data notshown). This occurs because, during the PCR process, DNA polymeraseextends the replicated strand past the position of the second primerinto adjacent repetitive sequences if the initial template containsgenomic DNA sequences. These extension products which are longer thanthe amplification product, are present in all such PCR reactions. Since,in the present method, repetitive sequences are adjacent to the segmentsbeing amplified, the extension products are likely to contain suchsequences. Phenol-chloroform extraction of PCR reactions does not removesuch extension products. PCR reaction mixtures containing thesesequences may hybridize to repetitive genomic DNA in addition to thetarget sequence. Hence, isolation of the purified genomic amplificationfragment (whether it is obtained directly from genomic DNA or by PCR),is a preferred embodiment of the subject invention and would not beobvious to one skilled in the art.

[0069] Insertion of the purified fragments into plasmids,bacteriophages, or artificial chromosome cloning vehicles capable ofbeing propagated in E. coli, yeast, or other species may be desirable toreduce the cost and labor required for repeated preparation of singlecopy DNA probes. A variety of cloning vectors have been optimized forrapid ligation and selection for vectors containing PCR products (forexample: U.S. Pat. Nos. 5,487,993 and 5,766,891). If the probe will beused in multiple hybridizations, then the cloned recombinant form willbe less expensive to produce in large quantities than by iterative PCRamplification from the same genomic DNA template. In addition, genomicinsert in the cloned probe does not have to be isolated duringpurification, since the fragment recombined with vector is propagated inthe absence of any other genomic DNA that could potentially containrepetitive sequences. Finally, the cloned vehicle provides a potentiallyinexhaustible source of probe, whereas natural genomic DNA templates mayhave to be reisolated from cell lines or from other sources. Single copyDNA fragments obtained by PCR amplification as described above areisolated according to size by gel electrophoresis and purified bycolumns as is well known in the art.

[0070] These fragments are then labeled with nonisotopic identifyinglabel such as a fluorophore, an enzymatic conjugate, or one selectedfrom the group consisting of biotin or other moieties recognized byavidin, streptavidin, or specific antibodies. There are several types ofnon-isotopic identifying labels. One type is a label which is chemicallybound to the probe and serves as the means for identification andlocalization directly. An example of this type would be a fluorochromemoiety which upon application of radiation of proper wavelengths willbecome excited into a high energy state and emit fluorescent light. Theprobes can be synthesized chemically or preferably be prepared using themethods of nick-translation (Rigby et al., J. Mol Biol., 113:237-251,(1977)) or Klenow labeling (Feinberg et al., Anal. Biochem.,137:266-267, (1984)) in the conventional manner using a reactantcomprising the identifying label of choice (but not limited to)conjugated to a nucleotide such as dATP or dUTP. The fragments areeither directly labeled with a fluorophore-tagged nucleotide orindirectly labeled by binding the labeled duplex to afluorescently-labeled antibody that recognizes the modified nucleotidethat is incorporated into the fragment as described below.Nick-translations (100 μl reaction) utilize endonuclease-free DNApolymerase I (Roche Molecular Biochemicals, Indianapolis, Ind.) andDNase I (Worthington Biochemical Corporation, Lakewood, N.J.). Eachfragment is combined with DNA polymerase (20 units/microgram DNA), DNaseI (10 microgram/100 μl reaction), labeled nucleotide (0.05 mm final) andnick translation buffer. The reaction is performed at 15 ° C. for 45minutes to 2 hours and yields a variety of labeled probe fragments ofdifferent nucleotide sizes in the 100 to 500 bp size range.

[0071] Other methods for labeling and detecting probes in common use maybe applied to the single-copy DNA probes produced by the present method.These include: fluorochrome labels (which resolve labeling on individualchromatids which serves as an affirmation that hybridization occurredunequivocably, and further allows detection precisely at site ofhybridization rather than at some distance away), chemical reagentswhich yields an identifiable change when combined with the properreactants (for example, alkaline phosphatase, horseradish peroxidase andgalactosidase, each of which reacts and provide a detectable colorchange that identifies the presence and position of the targetsequence), and indirect linkage mechanism of specifically bindingentities (such as the biotin-avidin system in which the probe ispreferably joined to biotin by conventional methods and added to anavidin- or streptavidin-conjugated fluorochrome or enzyme which providesthe specificity for attaching the fluorochrome or enzyme to the probe).

[0072] It will be recognized that other identifying labels may also beused with the described probes. These include fluorescent compositionssuch as energy transfer groups, conjugated proteins, antibodies andantigens, or radioactive isotopes.

[0073] Chromosomal hybridization and detection are a preferred use ofDNA probes generated by the present invention. DNA probes generated bythe present method may be hybridized either directly to complementarynucleic acids in cells (in situ hybridization) or to nucleic acidsimmobilized on a substrate. A preferred use of the method is in situhybridization, which is well known in the art, being described in U.S.Pat. Nos. 5,985,549; 5,447,841; 5,756,696; 5,869,237. Based on earlywork of Gall and Pardue (Proc. Natl. Acad. Sci., 63:378-383, 1969),isotopic in situ hybridization was established in the 1970s (see Gerhardet al., Proc. Natl. Acad. Sci., 78:3755-3759, 1981 and Harper et al.,Proc. Natl. Acad. Sci., 78:4458-4460, 1981 as examples) and subsequentlynonisotopic in situ hybridization was established. The technique ofnonisotopic in situ hybridization is reviewed and a protocol is providedfor use in chromosomal hybridization by Knoll and Lichter, in CurrentProtocols in Human Genetics, Vol. 1, Unit 4.3 (Green-Wiley, New York,1994) and in U.S. Pat. No. 5,985,549. The technique relies on theformation of duplex nucleic acid species, in which one strand is derivedfrom a labeled probe molecule and the second strand comprises the targetto be detected. Target molecules may comprise chromosomes or cellularnucleic acids. Numerous methods have been developed to label the probeand visualize the duplex.

[0074] The method of the present invention is intended to be used withany nucleic acid target containing repetitive sequences. The samplecontaining the target nucleic acid sequence can be prepared fromcellular nuclei, morphologically intact cells (or tissues), chromosomes,other cellular material components, or synthetically produced nucleicacids. The samples may be obtained from the fluids or tissues of amammal, preferably human, which are suspected of being afflicted with adisease or disorder either from a biopsy or post-mortem, or from plants.

[0075] As an example, chromosomal preparations can be made in thefollowing manner: phytohemagglutinin-stimulated peripheral lymphocytesare cultured in RPMI 1640 medium containing 10% fetal calf serum for 72hours at 37° C. Ethidium bromide (100 ug/10 ml final) is added 1-½ hoursprior to harvest. Colcemid (1 ug/10 ml final) is added during the final20 min of incubation with ethidium bromide. The cells are then pelletedby centrifugation and incubated preferably in 0.075 M KCl at 37° C. forabout 20 minutes. Cells are then pelleted again and fixed in 3 changesof Camoy's fixative (3:1 methanol:acetic acid volumetric ratio) usingconventional cytogenetic techniques. [For a review of chromosomepreparation from peripheral blood cells, see Bangs and Donlon inDracopoli et al., eds., Current Protocols in Human Genetics, Vol. 1,Unit 4.1 (Green-Wiley, New York, 1994)]. The nuclei or cells insuspension can then be dropped onto clear glass coverslips or microscopeslides in a humid environment to promote chromosome spreading. Thecoverslips or microscope slides can then be preferentially air driedovernight, aged or stored until required for use in in situhybridization.

[0076] Immediately prior to chromosomal denaturation in the in situhybridization procedure, the dried or stored chromosome preparations canbe pretreated in prewarmed 2×SSC (components are in Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual [Cold Spring HarborLaboratory Press, 1989]) for 30 minutes at 37° C. followed bydehydration in an ethanol series (2 minutes each in 70%, 80%, 90% and100% ethanol).

[0077] When the target nucleic acid sequence is DNA, DNA in the samplecan be denatured by heat or alkali. [See Harper et al., Proc. Natl.Acad. Sci., 78:4458-60 (1981), for alkali denaturation and Gall et al.,Proc. Natl. Acad. Sci., 63:370-383 (1969)]. Denaturation is carried outso that the DNA strands are separated with minimal shearing, degradationor oxidation.

[0078] In the preferred current method, the labeled single copy probe isresuspended in deionized formamide and denatured at 70-75° C. Thechromosomal template is denatured in a solution containing 70%formamide/2×SSC, pH 7.0 followed by dehydration in an ethanol series (2minutes each in cold 70% ethanol and room temperature 80, 90 and 100%ethanol). Hybridization of the labeled probe to the correspondingtemplate is carried out in a solution containing 50% formamide/2×SSC/10%dextran sulfate/BSA [bovine serum albumin; 1 mg/ml final] for a fewhours to overnight. The length of time depending on the complexity ofthe probe that is utilized. After hybridization, non-hybridizing excessprobe is removed by a washing procedure. The duplexes are treated with aseries of 15-30 minute washes: first with a solution of 50%formamide/2×SSC at 39-45° C., then 2×SSC at 39-45° C., followed by a15-30 minute wash at room temperature in 1×SSC. The hybridized sequencesare detected by relevant means. For example, digoxigenin-dUTP can be butis not limited to detection by an antibody to digoxigenin such asrhodamine or fluorescein conjugated antibody (Roche MolecularBiochemicals, Indianapolis, Ind.). Following detection, spuriousdetection reagents are removed by washing in varying SSC andSSC/triton-X concentrations, the chromosomes are counterstained with adye such as DAPI and the hybridized preparation is mounted in anantifade solution such as Vectashield (Vector Laboratories, Burlingame,Calif.). The cells are examined by fluorescence microscopy with theappropriate filter sets and imaged with a charge coupled device (CCD).

[0079] An important aspect of the present invention is that the probe ortarget DNA does not require pre-reaction with a non-specific nucleicacid competitor such as purified repetitive DNA or that the probe doesnot require experimental verification that the single copy fragments orrecombinant cloned probes do not contain repetitive sequences (U.S. Pat.Nos. 5,985,549; 5,447,841; 5,663,319; 5,756,696) because the probes aresingle copies without repetitive elements. This results in asignificantly improved signal to noise ratio. A signal to noise ratio isdefined as a ratio of the probability of the probe detecting a bona fidesignal of hybridization of the target nucleic acid sequence to that ofthe probability of detecting the background caused by non-specificbinding of the labeled probe.

[0080] The hybridization reactions carried out using the probes of theinvention are themselves essentially conventional. As indicated, twoexemplary types of hybridizations are the Southern blot and FISHtechniques, well known to those skilled in the art. However, the visualpatterns resulting from use of the probes, termed indicator patterns,are extremely useful tools for cytogenetic analyses, especiallymolecular cytogenetic analyses. These indicator patterns facilitatemicroscopic and/or flow cytometric identification of normal and abnormalchromosomes and characterization of the genetic abnormalities. Sincemultiple compatible methods of probe visualization are available, thebinding patterns of different components of the probes can bedistinguished, for example, by color. Thus, the invention is capable ofproducing virtually any desired indicator pattern on the chromosomesvisualized with one or more colors (a multi-color indicator pattern)and/or other indicator methods.

[0081] Preferred indicator patterns derived from using the probes of theinvention comprise one or more “bands,” meaning a reference point in agenome comprising a target DNA sequence with a probe bound thereto, andwherein the resulting duplex is detectable by some indicator. Dependingon hybridization washes and the detection conditions, a band can extendfrom the narrow context of a sequence providing a reliable signal to asingle chromosome region to multiple regions on single or pluralchromosomes. The indicator bands from the probes hereof are to bedistinguished from bands produced by pretreatment and chemical staining.The probe-produced bands of the present invention are based upon thecomplementarity of the DNA sequences, whereas bands produced by chemicalstaining depend upon natural characteristics of the chromosomes (such asstructure or protein composition), but not by hybridization to the DNAsequences thereof. Furthermore, chemical staining techniques are usefulonly in connection with metaphase chromosomes, whereas theprobe-produced bands of the present invention are useful for bothmetaphase and interphase chromosomes.

[0082] The following examples set forth the preferred techniquesemployed for the development, generation, labeling and use of specificDNA probes designed to hybridize to a target DNA sequence in a genome.It is to be understood, however, that these examples are provided by wayof illustration and nothing therein should be taken as a limitation uponthe overall scope of the invention.

EXAMPLE 1 Development of HIRA Gene Probe

[0083] A known genetic disorder on human chromosome 22 involves adeletion of one HIRA gene in chromosome band 22q11.2, i.e., in normalindividuals, there are two copies of the HIRA gene, whereas in affectedindividuals, only one copy is present. This deletion is considered to bea cause of haploinsufficiency syndromes such as DiGeorge andVelo-Cardio-Facial Syndromes (VCFS), because insufficient amounts ofgene product(s) may disrupt normal embryonic development (Fibison etal., Amer. J. Hum. Genet., 46:888-95 (1990); Consevage et al., Amer. J.Cardiol., 77:1023-1205 (1996)). Other syndromes including Cat EyeSyndrome and derivative chromosome 22 syndrome result from an excess ofgenomic sequences from this region (Mears et al., Amer. J. Hum. Genet.,55:134-142 (1994); Knoll et al., Amer. J. Med. Genet., 55:221-224(1995)). Typically individuals with these syndromes have supernumeraryderivative chromosome 22s.

[0084] Initially, a computer-based search using the search term “HIRA”was performed using Entrez Nucleotide software at the National Libraryof Medicine website. This identified a series of cDNA sequences for theHIRA gene in GenBank. The full length cDNA sequence was selected(GenBank Accession No. X81844), having 3859 bp. This cDNA sequence wasthen compared with the genome sequence which included draft sequences atthe National Library of Medicine(http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).This was done in order to determine whether genomic sequences ofsufficient length were available for probe development. This comparisonconfirmed that the entire HIRA genomic sequence was known, and that thecoding sequence interval spanned a length of 100,836 bp in thechromosome. Since the available contiguous genomic sequence in GenBankexceeded the length of the coding interval, it was possible to select aninterval longer than the coding region in order to include sequencesfrom the gene promoter at the 5′ end and untranslated sequences andpolyadenylation signal at the 3′ end. A total genomic interval ofapproximately 103 kb was thus selected. Position 1 of this 103 kbinterval corresponds to position 798,334 in GenBank Accession numberNT_(—)001039.

[0085] In the next step, the selected 103 kb genomic interval wascompared with known high-complexity repeat sequence family members orconsensus sequences that are aligned with the test genomic sequences(SEQ ID Nos. 1-428) and all combinations of low-complexity tandem repeatsequences of at least 17 nucleotides in length (mono-, di-, tri-, andtetranucleotide units) known to be present in the human genome (SEQ IDNos. 447-479). This comparison was done using the publicly availableCENSOR program which can be found at the Genetic Information ResearchInstitute website, www.girinst.org. This program utilizes theSmith-Waterman global alignment comparison algorithm to determine thelocations and distribution of repeat sequences within the genomicinterval. A Smith-Waterman alignment of repetitive with genomicsequences was performed with the following parameters: Length of marginsequence: 50 nt, minimum length to extract insertion: 12 nt, minimummargin to combine matching fragments: 30, similarity threshold: 22,similarity threshold to always keep match: 35, ratio threshold: 2.8,relative similarity threshold: 2.8, gap constant D1: 2.95, gap constantD2: 1.90, and mismatch penalty: −1.0. This analysis generated thefollowing table, which details the coordinates of repetitive sequencefamily members found in and adjacent to the human HIRA gene codingsequence. Position (bp) in Seq. Listing HIRA position Corresponding (bp)to HIRA Match Begin End Repeat Family SEQ ID NO. Begin End 798411 798434(AC) 452 1 24 798983 799395 MLT2A1 444 1 434 801257 801348 CHESHIRE_A420 132 223 801367 801729 L1ME_ORF2 425 757 1089 801746 802032 Alu-Jb 22 289 802033 802308 L1ME_ORF2 425 1090 1380 802355 802434 L1MB6_5 771629 1710 802448 802798 L1M3D_5 66 996 1348 802811 803100 Alu-Y 2 1 290803104 803189 L1M3D_5 66 907 995 803199 803454 Alu-Jb 2 5 290 803472803545 Alu-Spqxz 2 2 76 803548 804061 L1MEC_5 345 1860 2392 804079804365 Alu-Sz 2 6 290 804476 804559 L1P_MA2 348 6242 6321 804625 804885L1ME_ORF2 425 2287 2568 804936 804997 MLT1E2 106 198 260 805011 805077MLT1E1 105 420 484 805110 805211 L1PBA_5 359 103 204 805212 805862L1PBA_5 359 1089 1738 805933 805989 Alu-J 2 234 290 805991 806489L1PBA_5 359 1749 2247 806510 806624 L1 59 1659 1773 806628 806917 Alu-Sz2 1 290 806919 807254 L1M2_5 61 2377 2716 807301 808176 L1P_MA2 348 35164425 808179 808469 Alu-Sz 2 1 290 808476 808734 L1 59 3268 3525 808735809426 L1ME_ORF2 425 1411 2105 809429 809860 L1P_MA2 348 5607 6044809861 809993 Alu-Jb 2 2 134 809996 810282 Alu-Jb 2 2 290 810345 811040L1 59 4711 5402 811041 811221 L1PB3 358 151 333 811226 811513 Alu-Sx 2 1287 811515 812032 L1PB1 357 330 863 812096 812394 Alu-Jb 2 1 288 812474812698 Alu-Jb 2 5 229 812721 812836 Alu-Jo 2 2 117 812862 812901 L1P_MA2348 4315 4354 812903 813078 L1 59 3028 3222 813079 814102 L1ME_ORF2 4251113 2166 814323 814410 MER1B 315 242 337 814411 814557 CHARLIE3 7 1 281814780 814916 L1MB7 78 9 143 815061 815181 Alu-Y 2 1 134 815420 815452LTR67 279 99 131 816487 816772 Alu-Sx 2 5 290 817180 817270 L1MCC_5 3351384 1473 817332 817620 Alu-Sg 2 1 290 817634 817909 Alu-Sq 2 1 288817943 818227 Alu-Sx 2 2 289 818368 818578 HAL1 18 1346 1547 818631818791 LINE2 362 2280 2464 818824 818889 Alu-S 2 223 290 818890 819185LINE2 362 2465 2749 819328 819450 LINE2 362 1925 2049 819565 819757LINE2 362 2273 2498 823604 823892 Alu-Jo 2 2 290 826836 827042 Alu-Sxzg2 84 290 827922 827977 MIR 99 105 160 830762 831371 L1MEC_5 345 14982123 831396 831685 Alu-Sx 2 2 290 831687 831774 L1MEC_5 345 2117 2205831778 832066 Alu-Sx 2 1 290 832155 832288 Alu-FLA 2 5 134 832317 832431L1MC2 79 666 786 832442 832735 Alu-Sz 2 1 289 832742 832992 L1MC2 79 7871077 833004 833170 L1ME_ORF2 425 172 340 833177 834590 TIGGER1 148 11477 834592 834642 Alu-Jb 2 156 207 834799 834877 Alu-Jb 2 208 290834907 835194 Alu-Y 2 1 289 835198 835590 TIGGER1 148 1468 1900 835597835888 Alu-Sx 2 1 290 835946 835979 L1P_MA2 348 4654 4689 836060 836177MER2 316 229 345 836203 836486 Alu-Sx 2 7 290 836497 836712 MER2 316 1228 838478 838760 Alu-Sz 2 1 288 838822 839069 Alu-Sx 2 1 288 839086839373 Alu-Sz 2 1 289 840297 840926 L1MB7 78 269 915 841062 841306 L1MB778 7 249 841323 841382 L1ME_ORF2 425 3053 3116 841408 841697 Alu-Sq 2 1290 841705 841828 Alu-Jo 2 1 136 841829 842012 L1ME_ORF2 425 2870 3052842744 842871 MER86 239 51 183 842879 843107 Alu-Spqxz 2 3 230 843109843271 Alu-Jo 2 9 175 847056 847210 MER104 293 1 179 847256 847351 L1ME4343 128 224 847413 847551 MIR 99 65 218 847570 847695 L1ME4 343 1 127847865 848137 Alu-Y 2 1 290 848171 848458 Alu-Sg 2 1 290 848493 848564L1PA7 355 35 105 848646 848928 Alu-Sc 2 5 290 849186 849435 L1ME_ORF2425 2527 2796 849450 849745 Alu-Sx 2 5 289 850114 850249 L1P_MA2 3485447 5610 850250 850761 L1 59 3478 4017 850824 850942 L1ME_ORF2 425 11281265 851588 851614 (T) 449 1 27 (complement) 851749 851881 L1ME2 341 357523 852607 852853 L1MA10 72 664 918 852863 853156 Alu-Sc 2 1 290 853176853211 L1MA10 72 628 663 853212 853267 L1MA9 75 987 1041 853491 853779Alu-Sz 2 1 290 859137 859435 Alu-Sx 2 1 290 859436 859456 (A) 449 1 21859570 859805 L1ME3A 342 215 442 859806 860289 L1ME2 341 375 879 860318860605 Alu-Y 2 1 290 862194 862481 Alu-Sg 2 1 290 865060 865350 Alu-Sq 21 290 867521 867800 Alu-Jb 2 1 288 867836 867876 MIR 99 157 196 869546869802 LINE2 362 123 413 869923 870118 LINE2 362 1251 1450 870124 870202Alu-J 2 48 132 870203 870296 LINE2 362 1451 1592 870316 870666 LINE2 3621708 2097 871000 871075 LINE2 362 2617 2736 871650 871935 Alu-Jo 2 1 290871936 871960 (GAAAAA) 4 28 872154 872444 Alu-Sc 2 1 289 874867 874990L1MB7 78 529 676 878120 878408 Alu-Sx 2 1 290 881003 881054 MLT1G 109217 268 881130 881266 MLT1G 109 269 480 881293 881346 MLT1G 109 415 469881762 881891 LINE2B 363 85 229 882448 882740 Alu-Sb0 2 1 290 883566883716 Alu-Sz 2 1 288 883782 883977 Alu-Sc 2 2 290 883988 884329 L1P_MA2348 5600 5935 884333 884623 Alu-Sp 2 1 290 884624 885134 L1ME_ORF2 4252431 2975 885160 885456 Alu-Jb 2 9 290 885460 885742 L1ME_ORF2 425 29493252 885744 886031 Alu-Sx 2 1 288 886032 886082 Alu-Sp 2 291 341 886083886166 L1MB7 78 137 220 886168 886454 Alu-Sc 2 1 290 886535 887059 L1MB778 345 901 887169 887460 Alu-Y 2 1 289 887485 887748 L1MD2 337 794 1072887752 887779 LOR1I 366 395 422 888253 888318 LINE2 362 2440 2505 888385888548 LINE2 362 2579 2739 888865 888893 LOR1I 366 394 422 889006 889296Alu-Jb 2 5 290 889446 889548 Alu-Jo 2 188 290 889549 889677 L1PB3 358770 897 889842 890133 Alu-Sq 2 1 290 890515 890797 Alu-Sz 2 1 283 890858890972 L1ME2 341 769 885 890986 891024 LOR1I 366 396 434 891028 891063LTR66 266 173 207 891126 891536 LINE2 362 1980 2452 891545 891670LTR16A1 382 9 128 891688 891963 LTR16A 381 146 429 892907 893013 LINE2362 2636 2747 893851 893924 MLT1L 119 47 119 894528 894849 Alu-Sx 2 1290 895825 895903 LINE2 362 2592 2664 895912 896083 MER20 317 46 216897067 897299 MER20 317 2 217 897492 897624 Alu-FLA 2 2 136 897977898261 Alu-Sc 2 1 290

[0086] The lengths of the non-repetitive intervals were calculated fromthese data. For example, a non-repetitive interval of 5358 bp wasdetermined between coordinate positions 853779 and 859137 whichdelineate the boundaries of adjacent Alu-Sz and Alu-Sx repetitiveelements. Next, the non-repetitive intervals were sorted based on theirrespective lengths. Four of these non-repetitive intervals were selectedfor probe development, namely the above-referenced 5358 bp sequence, a3847 bp sequence (coordinates 819757 and 823604), a 3785 bp sequence(coordinates 843271 and 847056), and a 3130 bp sequence (coordinates874990 and 878120).

[0087] In the next step, the long PCR technique was used to amplifyportions of the four identified single copy intervals. The techniquefollowed for amplification of the 5358 bp interval is described indetail below. Similar techniques were followed for amplification of theremaining three single copy intervals.

[0088] Probes of maximal length were desired for FISH experiments.However, in order to optimize the PCR reaction that generated theseprobes, other constraints had to be met, which resulted in amplificationproducts somewhat shorter than the entire non-repetitive sequenceinterval. The Prime computer program was employed to optimize theselection of primers for PCR (Genetics Computer Group software package,Madison Wis.). The PCR primers which were optimized for long PCR wereconstrained as follows: size of 30-35 nucleotides; GC content of 50-80%;melting temperature of 65-70° C.; the primer was not permitted toself-anneal at the 3′ end with hairpins of greater than 8 nucleotides;the primer was not permitted to self-anneal at any position with greaterthan 14; and the primer was permitted to anneal only at a singleposition in the target sequence and primer-primer annealing was limitedat the 3′ end to less than 8 bp and at any other point less than 14 bp.In addition, certain constraints were applied to the amplified PCRproduct in order to optimize long PCR: length of 5100-5358 nucleotides;GC content of 40-60%; melting temperature of 70-95° C.; difference inforward and reverse primer melting points less than 2° C. This yielded apossible 517 forward primers and 382 reverse primers, and a total of 928possible products. The Prime program using the foregoing constraintsrank ordered potential primer pairs. The top ranked primers wereselected for synthesis, as set forth in the following Table 2. Theseprimers were commercially produced (Oligos, Etc., Wilsonville, Oreg.).TABLE 2 GenBank Accession Coordinates of Longest Forward PCR PrimerReverse PCR Primer PCR Primer SEQ ID Probe Chromosome No., ChromosomeSingle Copy Intervals, Coordinates, Coordinates, Nos., Length Gene BandGenomic Sequence Beginning/End Beginning/End Beginning/EndForward/Reverse (bp) HIRA 22q11.2 NT_001039 853779/859137 853946/853975859116/859085 429/430 5170 HIRA 22q11.2 NT_001039 819757/823604819901/819933 823592/823559 431/432 3691 HIRA 22q11.2 NT_001039843271/847056 843602/843631 846946/846915 433/434 3344 HIRA 22q11.2NT_001039 874990/878120 875226/875257 878074/878042 435/436 2848

[0089] Using these primers, a long PCR reaction (50 μl) was performedusing 1 microgram of high molecular weight genomic DNA (purified byphenol extraction) and 200 μM of each oligonucleotide primer to amplifythe 5170 bp probe. Specifically, high fidelity DNA polymerase (LA-Taq,Takara Chemical Co.) was employed using the following thermal cyclingprotocol:

[0090] Step 1—94° C.—5 minutes

[0091] Step 2—98° C.—20 seconds

[0092] Step 3—65° C.—7 minutes

[0093] Step 4—14 times to Step 2

[0094] Step 5—98° C.—20 seconds

[0095] Step 6—65° C.—7 minutes +15 s/cycle

[0096] Step 7—14 times to Step 5

[0097] Step 8—72° C.—10 minutes

[0098] Step 9—0° C.

[0099] Step 10—END

[0100] Because amplification of the 5170 bp probe is less efficient thanamplification of shorter fragments, the initial PCR reaction did notyield sufficient quantities of probe for multiple hybridizationexperiments. Therefore, a 4 μl aliquot of the original DNA amplificationreaction was reamplified using the following protocol: Step 1—94° C.—1.5minutes, followed by Steps 2-10 of the original PCR reaction. Sufficientquantities of the 5170 bp probe were obtained. An alternative toreamplification is to increase Step 7 by at least 10 cycles.

[0101] The amplified product was then purified by gel electrophoresisfollowed by column chromatography. First, the amplified product wasseparated on a 0.8% Seakem LE agarose gel (FC Bioproducts) in 1×modifiedTAE buffer. The gel was then stained with ethidium bromide andvisualized with UV light. The fragment corresponding to the correctinterval size was excised in an Ultrafree-DA spin column (Millipore) andcentrifuged at 5000 g for 10 minutes. The DNA was recovered in solutionand precipitated in {fraction (1/10)} V NaOAc and 2.5 V 95% EtOH(overnight) at −20° C. The precipitated DNA was then centrifuged, rinsedwith cold 70% EtOH, air dried and resuspended in 20 μl of steriledeionized water. The DNA was checked on a 0.8% agarose gel (Sigma) todetermine DNA concentration.

[0102] The detailed probe labeling, hybridization, removal ofnon-specifically bound probe, and probe detection procedures aredescribed by Knoll and Lichter, In: Dracopoli et al., (eds), “CurrentProtocols in Human Genetics Volume 1”, Unit 4.3 (Green-Wiley, New York,1994). Briefly, in order to label the probe, a standard nick translationreaction was carried out (Rigby et al., J. Mol. Biol., 113:237-251,(1977)) using digoxigenin-11-dUTP as the label. This yielded a series ofoverlapping 300-500 bp labeled fragments, which together comprised the5170 bp probe.

[0103] The labeled probe fragments were then precipitated by adding{fraction (1/10)} V NaOAc plus 2.5 V 95% EtOH and carrier DNA(overnight, −20° C.). On the following day, the precipitated DNA wascentrifuged, lyophilized, and resuspended in deionized sterile water ata concentration of 125 ng/20 μl.

[0104] A comparison set of hybridizations were carried out with normaldenatured human metaphase chromosomes, using the labeled probe fragmentswith and without blocking nucleic acid of the type described in U.S.Pat. Nos. 5,447,841, 5,663,319 and 5,756,696. Twenty μl of resuspendedlabeled probe was then lyophilized and resuspended in 10 μl of deionizedformamide and denatured for 5 minutes at 70-75° C. to yieldsingle-stranded nucleic acids. For comparison, probes were pre-reactedwith purified repetitive DNA by adding 125 ng (or 20 μl) of labeledprobe to 10 micrograms of C₀t 1 DNA (Life Technologies) and lyophilizingthe mixture. This mixture was then denatured for 5 minutes at 70° C.followed by pre-reaction (or pre-annealing) for 30 minutes at 37° C. toconvert the single stranded repetitive sequences in the probe to doublestranded nucleic acid. This disables the hybridization between thesequences and the chromosome as target DNA template.

[0105] Subsequently, the denatured probes with or without purifiedrepetitive DNA (i.e., C₀t 1) were mixed with 1 V prewarmed hybridizationsolution (comprised of 4×SSC/2 mg/ml nuclease free bovine serumalbumin/20% dextran sulfate/30% sterile deionized water) and overlaidonto denatured target DNA. The chromosomal target DNA, fixed to amicroscope slide had been denatured at 72° C. for 2 minutes in 50%formamide/2×SSC. A coverslip was placed over the probe hybridizationmixture on the slide, sealed with nail polish enamel to preventevaporation and placed in a moist chamber at 39° C. overnight.

[0106] Following hybridization, non-specifically bound probe was washedoff with varying stringencies of salt concentration and temperature. Thelabeled probes, pre-reacted to disable repetitive sequencehybridization, and the probes without such pre-reaction were detectedwith rhodamine-labeled antibody to digoxigenin-11-dUTP, using aconventional FISH protocol (Knoll and Lichter, Current Protocol in HumanGenetics, Vol. 1, Unit 4.3, Green-Wiley, New York, 1994). ChromosomalDNA was counter-stained with DAPI. The cell preparations on microscopeslides were then mounted in antifade solution (such as Vectashield,Vector Laboratories, Burlingame, Calif.) and visually examined using afluorescence microscope with the appropriate fluorochrome filter sets.FIGS. 1 and 2 are photographs illustrating the results of thecomparative hybridizations, where FIG. 1 is the hybridization with theblocking repetitive sequences, while FIG. 2 is the hybridization withoutpre-reaction with purified repetitive DNA. These photographs depicthybridization to both HIRA alleles on two normal chromosome 22q11.2regions. A comparison of the photographs demonstrates that the presenceof the blocking repetitive sequences is unnecessary using the probes ofthe present invention.

[0107] The remaining three probes identified in Table 2 werePCR-amplified and labeled as described above. These probes were used ina series of FISH experiments to determine the efficacy of the probes.Thus, all four probes were used together without pre-annealing ofpotentially repetitive sequences (FIG. 6), and a combination of thethree shortest probes were used on cells from a patient affected withDiGeorge/VCFS with a previously confirmed deletion (FIG. 12). In theFIG. 6 photograph, the probe was hybridized to a single region of bothchromosome 22s in a normal individual (arrows) In FIG. 12, only onechromosome 22 hybridized (arrow). The other chromosome 22, as indicatedby a star, has a deletion of this region and does not hybridize to theprobe.

EXAMPLE 2 Development of NECDIN and CDC2L1 Gene Probes

[0108] The techniques described in Example 1 were used to develop aseries of probes for detecting known genetic disorders on chromosome 1(Monosomy 1p36.3 syndrome; Slavotinek et al., J. Med. Genet., 36:657-63(1999)) and on chromosome 15 (Prader-Willi and Angelman Syndromes).Approximately 70% of patients with Prader-Willi or Angelman syndromeexhibit hemizygous deletions of the sequence containing the NECDIN gene(Knoll et al., Amer. J. Med. Genet., 32:285-290 (1989); Nicholls et al.,Amer. J Med. Genet., 33:66-77 (1989)). The presence of excess copies ofthis gene is diagnostic for an abnormal phenotype in patients withinterstitial duplication or a supernumerary derivative or dicentricchromosome 15 (Cheng et al., Amer. J. Hum. Genet., 55:753-759, (1994);Repetto et al.,Am. J. Med. Genet., 79:82-89, (1998)). The followingTable 3 sets forth the deduced single copy intervals, PCR primercoordinates, SEQ ID Nos., and the lengths of the resultant probes. TABLE3 GenBank Accession Coordinates of Longest Forward PCR Primer ReversePCR Primer PCR Primer SEQ ID Probe Chromosome No., Chromosome SingleCopy Intervals, Coordinates, Coordinates, Nos., Length Gene Band GenomicSequence Beginning/End Beginning/End Beginning/End Forward/Reverse (bp)CDC2L1¹ lp36.3 AL031282  8823/17757 9137/9167 13960/13931 444/443 4823CDC2L1¹ lp36.3 AL031282  8823/17757 13028/13057 17752/17720 445/446 4724NECDIN 15q11-q13 AC006596 94498/99152 94501/94535 98567/98601 439/4404166 NECDIN 15q11-q13 AC006596 68031/75948 72122/72156 75666/75637437/438 3544 NECDIN 15q11-q13 AC006596 76249/79221 76608/7663978898/78867 441/442 2290

[0109] PCR-amplification was performed using the CDC2L1 primers in Table3, and products were labeled, hybridized and detected as set forth inExample 1. The labeled probes were used in a series of FISH experiments,with images of the hybridizations provided as FIGS. 7-10. In theexperiment shown in FIG. 7, the longest 4823 bp probe was employed andpotential hybridization repetitive sequences was disabled bypre-annealing with purified repetitive DNA. As a comparison, the sameprobe was used without pre-annealing of purified repetitive DNA (FIG.8). The hybridizations appear identical demonstrating that the presenceof purified repetitive DNA to block repetitive sequence hybridization isunnecessary. In both instances, the chromosomes with one or bothchromatids hybridized are indicated by arrows. In the experiments shownin FIGS. 9 and 10, the 4823 bp and 4724 bp probes were employed, with(FIG. 9) and without (FIG. 10) pre-annealing of the purified repetitiveDNA. Again, pre-reaction of the purified repetitive DNA is shown to beunnecessary using the probes of the invention.

[0110] The NECDIN probes were also used in a series of FISH experiments,as shown in FIGS. 3-5 and 11. These probes detected DNA sequencesbetween 36 and 62 kb distal of the NECDIN gene. The 3544 bp probe (SEQID Nos. 437-438) detected the 3′ terminus of the MAGEL2 gene. In FIG. 3,the 3544 bp probe was used on metaphase cells from a normal individual,with pre-annealing using purified repetitive sequences; FIG. 4 is acomparison, without pre-annealing. In FIG. 11, all three probes wereused in combination, on metaphase cells from a patient affected withPrader-Willi syndrome known to harbor a deletion of 15q11-q13 sequenceson one chromosome 15. The normal homolog is indicated by an arrow andshows hybridization to a single chromatid. The location of the deletedchromosome is indicated by a star. It does not show hybridization withthe probe.

[0111] The foregoing examples demonstrate that the mixed combinations ofDNA fragments give identical hybridization results, as compared with thefragments when used individually. This establishes that none of thefragments used individually or in combination will hybridize to anyother location in the genome and hence, are free of repetitivesequences. This provides an additional confirmation of the validity ofthe present method for the design and production of single copy genomicprobes.

[0112] Current use of commercial and research genomic probes to detectthese disorders requires that hybridization of repetitive sequences bedisabled prior to annealing of the probe to metaphase or interphasechromosomes. This increases the number of steps required to perform theprotocol and could potentially increase the chances of procedural errorsoccurring, any of which would be unacceptable in the clinical diagnosticlaboratory. The results present in FIG. 7 are comparable to thoseobtained using related commercially available genomic probes to detectthese abnormalities. Hence, these probes will be useful in the detectionof these genetic disorders. The probes themselves or in combination withother solutions necessary for hybridization and detections can beprovided to clinical laboratories as kits for detection of these geneticdisorders.

[0113] The probes developed from genomic sequences other than thosepresented as examples cited herein can also be utilized to detectinherited, sporadic, or acquired chromosomal rearrangements. Theserearrangements may correspond to numerous other known geneticabnormalities (including neoplasias) and syndromes besides thoseexamples given above. Hence, the present invention can also be usefulfor producing probes from genomic regions where no commercial probes areavailable or the probes are imprecise.

[0114] In principle, the present method can be utilized to design,develop and produce single-copy genomic probes for any genomic intervalwhere the DNA sequence is available and where a comprehensive set ofrepetitive sequence elements in the genome has been cataloged. Suchcatalogs are currently available for genomes for the following organisms(http://www.girinst.org): Homo sapiens, Mus musculus, Arabidopsisthaliana, Canorhabditis elegans, Drosophila melanogaster, and Daniorerio.

EXAMPLE 3

[0115] In this example, a number of probes specific to additionalgenetic disorders and cytogenetic abnormalities were developed using theprinciples of the invention. Software was also developed and improved toexpedite the process of designing single copy probes (findi.pl,prim_wkg, and prim, referred to above and provided on the accompanyingCD-R). The probes were subsequently tested and their utility confirmedby in situ hybridization.

[0116] Identification of Single Copy Sequences

[0117] The locations of single copy probe sequences are determineddirectly from long contiguous genomic DNA sequences. The locations weredetermined by software that aligns the sequences of repetitive sequencefamily members with the target genomic sequence. Comparison of thetarget sequence with previously determined sequences of repetitivefamily members served to identify and delineate the bounds of repetitiveelements within the target. The computer program, RepeatMasker(http://ftp.genome.washing-ton.edu/RM/RepeatMasker.html: Smit A. F. A. &Green P., unpublished results), was used to determine the locations ofrepetitive sequence families in contiguous genomic sequences, usually˜100 kb in length. RepeatMasker compares a genomic sequence with acompilation of repetitive sequence families present in multiple copiesin the human genome (http://www.girinst.org/server/repbase.html). Thisrepeat sequence database contains representative and consensus sequencesfor the majority of human repetitive sequence families. The database canbe expanded by addition of newly discovered repetitive sequence families(as shown in Example 6).

[0118] A Perl script (findi.pl) parsed the coordinates of the boundariesof the repetitive segments from RepeatMasker output, and then deducedand sorted the adjacent single copy intervals by size greater than aparametrized threshold (˜2 kb, in most instances). This scriptdetermines the locations and lengths of single copy intervals sorted bysize from the output file (with the suffix: out) produced byRepeatMasker, which contains a table of locations and lengths of repeatfamily elements. The boundaries of adjacent single copy intervals werededuced by subtracting one nucleotide position from the upstreamboundary of a repetitive element and adding one nucleotide position tothe downstream boundary of the previous element. Single copy intervalswith identical upstream and downstream coordinates (1 bp in length) wereconsidered to be adjacent repetitive sequences. Probe sequences werethen compared with the human genome sequence database (Altschul et al.,J. Mol. Biol., 215:403-410 (1990)) to determine if there was similarityto sequences elsewhere in the genome (such as duplicons or triplicons orother less well conserved intervals). Probe sequences that are weaklyconserved elsewhere in the genome do not cross-hybridize to thosetargets.

[0119] Oligonucleotide primers were selected for PCR amplification ofthe longest single copy intervals. A Unix wrapper script (prim_wkg)iteratively modifies the switches in the file containing the command todesign primers (prim), thus optimizing primer selection by changing thefollowing parameters for input to the program, Prime (Genetics ComputerGroup; Madison, Wis.): T_(m) (from 70-60° C.), G/C composition (from55-40%), and minimum interval length (from 90%-80% of the length of thesingle copy interval).

[0120] Probe Generation and Chromosomal in Situ Hybridization

[0121] DNA fragments were amplified by long PCR (Cheng et al., Proc.Natl. Acad. Sci. U.S.A., 91:5695-5699 (1994)) with LA-Taq as recommendedby the manufacturer (Panvera, Madison, Wis.). Other enzymes for long PCRhave been demonstrated to produce comparable results, including thosemanufactured by Roche Molecular Biochemicals, Indianapolis, Ind.;Stratagene, LaJolla, Calif.; and Invitrogen, Carlsbad, Calif. Theamplicons were purified by low-melt temperature agarose gelelectrophoresis, followed by chromatography with Micro-con 100 columns(Millipore, Bedford Mass.), which removed contaminating extensionproducts containing repetitive sequences.

[0122] Probe fragments were labeled by nick translation using modifiednucleotides such as digoxigenin-dUTP or biotin-dUTP (Roche MolecularBiochemicals, Indianapolis, Ind.). Labeled probes were denatured andhybridized to fixed chromosomal preparations on microscope slides usingpreviously described conditions (Knoll and Lichter, Current Protocols inHuman Genetics, Vol. 1, Unit 4.3, Green-Wiley, New York (1994)), withthe exception that preannealing of the probe(s) with repetitive DNA(such as C₀t1 DNA) was not utilized in a parallel set of hybridizations.Probes from a single chromosome region of 100 kb were hybridizedindividually or in combination to remove non-specific binding.Post-hybridization washes were performed at 42° C. in 50% formamide in2×SSC, followed by an additional wash at 39° C. 2×SSC and one in 1×SSCat room temperature. Wash stringency was increased, if necessary, toremove hybridization of probes to related sequences elsewhere in thegenome. Hybridized probes were detected with a fluorochrome (such asrhodamine or fluorescein) tagged antibody to the modified nucleotide.Chromosome identification was performed by counterstaining the cellularDNA with 4′,6-diamidino-2-phenylindole (DAPI). Hybridized chromosomeswere viewed with an epifluorescence microscope (Olympus, Melville, N.Y.)equipped with a motorized multi-excitation fluorochrome filter wheel.Hybridization patterns on at least 20 metaphases (and 50-100 nuclei)were scored for each probe or combination of probes, with and withoutpreannealing to C₀t1 DNA. Cells were imaged using a CCD camera (Cohu,Inc, San Diego, Calif.) and CytoVision ChromoFluor software (AppliedImaging, Santa Clara, Calif.).

[0123] Table 4 sets forth the data generated using the foregoingprocedure, with respect to a number of probes specific to knowndisorders and cytogenetic abnormalities. The abnormality designationmakes use of standardized nomenclature as set forth in ISCN 1995, AnInternational System for Human Cytogenetic Nomenclature (1995),Mittelman F, ed. TABLE 4 Representative Cytogenetic Abnormality Detectedby Probes on Metaphase Gene or Disorder Chromosomes Transcript IntervalAngelman Syndrome ish del(15)(q11.2q11.2)(UBE3A−) UBE3A IVS 8-IVS 9 andPrader-Willi ish del(15)(q11.2q11.2)(IC/SNRPN−) IC/SNRPN IVS 3′ to Exonu1B* Syndrome Duplication 15 ish dup(15)(q11.2q13)(UBE3A++, IC/SNRPN IVS5′-Exon u1B¹-IVS Syndromes IC/SNRPN++) and ish dic(15q11.2q13) 3′(UBE3A++, IC/SNRPN++) IC/SNRPN IVS 5′-Exon u1B Kallmann Syndrome ishdel(X)(p22.3)(KAL1−) KAL1  ˜53 kb downstream KAL1 IVS 10 Turner Syndromeish del(X)(p22.3)(SHOX−) SHOX IVS 1-Exon 2 and Distal Xp SHOX IVS 2Deletions SHOX IVS 3 ish del(X)(p22.3)(GS2−) GS2 Promoter-IVS 2 ishdel(X)(p22.3)(TBL1−) TBL1 IVS 2 TBL1 3′ UTR Down Syndromeish(21)(q22.2q22.3)(DSCR4 × 3) DSCR4²  ˜39 kb upstream DSCR4²  ˜30 kbupstream DSCR4²  ˜20 kb upstream Chronic Myelogenous ish t(9; 22)(q34;q11.2)(BCR st) BCR Proximal to the major Leukemia and breakpoint in CMLAcute Lymphoblastic BCR Proximal to the major Leukemia breakpoint in CMLBCR IVS 8 ish t(9; 22)(q34; q11.2)(ABL st) ABL1 Exon 1B-IVS 1B ABL1³ IVS1B ish t(9; 22)(q34; q11.2)(ABL mv) ABL1 IVS 4-IVS 6 ABL1 Exon 11-IVS 11ish t(9; 22)(q34; q11.2)(ABL mv) ABL1 IVS 3 ABL1³ IVS 3 ABL1 IVS 3 ABL1IVS 4-IVS 6 Williams Syndrome ish del(7)(q11.23q11.23)(LIMK1−) LIMK1 IVS13-3′UTR LIMK1 IVS2 Acute Myelogenous ish inv(16)(p13q22)(PM5 sp)PM5^(2,4)  ˜20 kb downstream Leukemia-Type M4 PM5^(2,4)  ˜60 kbdownstream ish inv(16)(p13q22)(PLA2G10 mv, PKD mv, PLA2G10⁴ IVS 3 PM5sp) PKD IVS 12-Exon 15 PM5 ˜100 kb upstream PLA2G10⁴ IVS 3 PKD Exon15-IVS 20 PM5 ˜100 kb upstream & ˜300 kb downstream ishinv(16)(p13q22)(ABCC1st) ABCC1 IVS 6 Rubinstein-Taybi ishdel(16)(p13.3)(CREBBP−) CREBBP IVS 18 Syndrome Acute Lymphocytic isht(12; 21)(p13.2; q22.1)(AML1 st) AML1 IVS 1-IVS 2 Leukemia ish t(12;21)(p13.2; q22.1)(TEL/ETV6 mv) TEL/ETV6 IVS 4 TEL/ETV6 IVS 3 TEL/ETV6IVS 2 Cri-du-Chat ish del(5)(p15.3)(CTNND2−) CTNND2 IVS 17 SyndromeCTNND2 IVS 14 ish del(5)(p15.3)(SEMA5A−) SEMA5A IVS 3 SEMA5A IVS 3SEMA5A IVS 3 ish del(5)(p15.3)(SLC6A3−) SLC6A3 IVS 3 Langer-Giedeon ishdel(8)(q23.3q24.1)(TRPS1−) TRPS1 IVS 1 Syndrome TRPS1 IVS 1Smith-Magenis ish del(17)(p11.2p11.2)(ADORA2B−) ADORA2B⁵ Promoter-IVS 1Syndrome ADORA2B⁵ IVS 1 ish del(17)(p11.2p11.2)(FLI1−) FLI1 IVS 9-IVS 12FLI1 IVS 12-IVS 14 FLI1 IVS 15-Exon 21 ish del(17)(p11.2p11.2)(MFAP4−)MFAP4 IVS 2-3′ UTR ish del(17)(p11.2p11.2)(ZNF179/PAIP1/ ZNF179-PAIP1-³Between ZNF179 and SHMT−) PAIP1. SHMT1 IVS 4 ishdel(17)(p11.2p11.2)(LGLL/HUGL−) LLGL Promoter-Exon 2 HUGL Promoter-IVS 1Charcot-Marie-Tooth ish dup(17)(p11.2p11.2)(PMP22++) PMP22 PromoterDisease Type  (˜5 kb upstream) 1A PMP22  ˜22 kb downstream PMP22⁵ IVS 3PMP22 IVS 3 Miller-Dieker ish del(17)(p13.3)(PAFAHIB1/EIF-3−) PAFAHIB1 ˜5 kb downstream Syndrome EIF-3⁶ IVS 24-IVS 27 PAFAHIB1 ˜7-8 kbdownstream EIF-3 IVS 15-IVS 19 PAFAHIB1  ˜13 kb downstream EIF-3 IVS5-IVS 11 Alagille Syndrome ish del(20)(p12.3p12.3)(JAG1−) JAG1 IVS 5-IVS8 JAG1 IVS 2-IVS 3 JAG1 Exon 1-IVS 2 Monosomy 13q32 ishdel(13)(q32.3)(ZIC2−) ZIC2  ˜5.8 kb downstream Trisomy 13ish(13)(q32.3)(ZIC2 × 3) ZIC2  ˜2 kb upstream Wolf-Hirschom ishdel(4)(p16.3)(HD−) HD Exon 67 Syndrome Sequence ID Forward PCR PrimerReverse PCR Forward/ GenBank Coordinate, Primer Coordinate, ReverseDisorder Accession No. Beginning/End Beginning/End Primers AngelmanSyndrome AC004600 41085/41119 45354/45325 480/481 and Prader-WilliAC004737 13740/13769 15414/15387 482/483 Syndrome Duplication 15AC004737 31102/31128 33347/33323 484/485 Syndromes AC004737 47792/4782149470/49441 486/487 Kallmann Syndrome AC006062 104433/104465107097/107072 488/489 AC006062 38822/38852 42042/42012 490/491 TurnerSyndrome NT_001151 44615/44646 47505/47473 492/493 and Distal XpNT_001151 49637/49669 52251/52217 494/495 Deletions NT_00115154357/54387 56821/56791 496/497 NT_001457 78970/79000 82994/82960498/499 NT_001159 175379/175409 179665/179633 500/501 NT_001159247264/247293 251290/251257 502/503 Down Syndrome AP000160 31007/3104132999/32965 504/505 AP000160 40725/40754 43078/43045 506/507 AP00016049973/50006 52409/52376 508/509 Chronic Myelogenous U07000 100745/100776104145/104115 510/511 Leukemia and U07000 74699/74728 77903/77874512/513 Acute Lymphoblastic U07000 114946/114978 117457/117426 514/515Leukemia U07561 27182/27213 29388/29357 516/517 U07562 9193/922211035/11004 518/519 U07563 65951/65985 70266/70237 520/521 U0756378862/78891 83813/83784 522/523 U07563 55807/55836 58077/58046 524/525U07563 53570/53604 55489/55455 526/527 U07563 55854/55888 57848/57817528/529 U07563 66333/66367 70295/70264 530/531 Williams SyndromeNT_000398 59947/59976 62211/62187 532/533 NT_000398 31966/3199335015/34989 534/535 Acute Myelogenous NT_000691 24509/24538 27988/27958536/537 Leukemia-Type M4 NT_000691 64204/64233 67682/67652 538/539NT_000691 68271/68300 71986/71957 540/541 NT_000691 71957/7198675481/75452 542/543 NT_025903 313783/313812 315675/315645 544/545Rubinstein-Taybi NT_000671 58833/58862 63347/63318 546/547 SyndromeAcute Lymphocytic AP000057 98712/98741 102903/102872 548/549 LeukemiaNT_000601 95456/95480 97283/97260 550/551 NT_000601 72543/7256474385/74361 552/553 NT_000601 38216/38245 40091/40062 554/555Cri-du-Chat NT_000149 169655/169685 171976/171945 556/557 SyndromeNT_000149 199168/199202 203507/203473 558/559 NT_000147 23905/2393527710/27676 560/561 NT_000147 30757/30790 33241/33209 562/563 NT_00014714716/14748 17787/17753 564/565 AF119117 28206/28239 31894/31860 566/567Langer-Giedeon NT_002886 267731/267760 270758/270724 568/569 SyndromeNT_002886 271242/271271 274437/274404 570/571 Smith-Magenis NT_00077056443/56472 58524/58491 572/573 Syndrome NT_000770 77442/7747579222/79189 574/575 U80184 6094/6127 7300/7267 576/577 U80184 7424/74538742/8708 578/579 U80184 9615/9647 11738/11704 580/581 NT_000760132621/132654 134663/134634 582/583 AL035367 9818/9850 12272/12241584/585 AL035367 1194/1226 5365/5334 586/587 Charcot-Marie-ToothAC005703 153173/153202 155027/154994 588/589 Disease Type AC005703215632/215661 217362/217329 590/591 1A AC005703 184666/184700186035/186006 592/593 AC005703 176746/176778 179073/179044 594/595Miller-Dieker NT_000774 63645/63679 66603/66573 596/597 SyndromeNT_000774 68841/68870 71195/71163 598/599 NT_000774 75328/7536278122/78093 600/601 Alagille Syndrome AL035456.24 153935/153966157675/157642 602/603 AL035456.24 144875/144904 147028/146995 604/605AL035456.24 135644/135673 139440/139407 606/607 Monosomy 13q32 AL355338111114/111145 116046/116012 608/609 Trisomy 13 AL355338 128595/128627133039/133006 610/611 Wolf-Hirschom NT_000102 267614/267643271120/271091 612/613 Syndrome

EXAMPLE 4

[0124] In this example, a more precise chromosomal breakpointdetermination was made using the probes of the invention. Structuralchromosome rearrangements can be inherited in genetic disease oracquired as in the case of certain cancers. They can occur within asingle chromosome (such as an inversion, deletion or duplication) orbetween homologous or non-homologous chromosomes (i.e. translocations).With the probes of the invention, the precise region of breakage can bedetermined at a previously unprecedented level of resolution. Suchresolution permits detection of genes or sequences that are disrupted inthe formation of the rearrangement and may provide insight intoetiology, prognosis and/or treatment. In inherited contiguous genesyndromes, precise localization of chromosome breakpoints can define theextent of deletion or duplication and hence the prognosis of thedisorder (Cheng et al., Am. J. Hum. Genet., 55:753-759 (1994)).

[0125] The following example illustrates how the single copy probeshereof provide more precise information than commercially availablecloned probes for the same chromosomal region.

[0126] In most cases of chronic adult myeloid leukemia (CML; 90%) and insome cases of acute lymphoblastic leukemia (ALL; 25-30% adults; 2-10%children) (Perkins et al., Cancer Genet. Cytogenet., 96(1): 64-80(1997); Rubintz et al., J. Pediatr. Hematol. Oncol, 20 (1):1-11(1998)),a reciprocal translocation between chromosome 9q34 and 22q11.2 isevident (Rowley, Nature, 243:290-392 (1973)). The abnormal or derivativechromosome 22 that results from this translocation fuses the ABL1oncogene on chromosome 9 to the BCR (breakage cluster region) promoteron chromosome 22. The ABL1 oncogene is expressed as either a 6 or a 7 kbmRNA transcript with alternatively spliced first exons, exons 1b and 1arespectively, spliced to the common exons 2-11. Exon 1b is ˜250 kbproximal of exon 1a and this very long intron is a primary target fortranslocations. In CML, the ABL1 gene is translocated from chromosome 9to the promoter of the BCR gene on chromosome 22 to produce a chimericBCR-ABL1 protein (Bernards, et al., Mol. Cell. Biol., 7:3231-3236;(1987). The BCR gene contains 24 exons and encodes a 160 kD protein. TheBCR breakpoints differ in CML and ALL. In CML, most breakpoints occurwithin the 5.8 kb major breakpoint cluster region (M-BCR) whichcorresponds to exons 12 through 17 (or b1 through b5); whereas in mostALL, the BCR gene breaks between exons 1 and 2 (minor or m-BCR). Thusdifferent molecular rearrangements resulting in differently-sizedproteins occur in each disorder. These rearrangements can bedistinguished with the probes of the invention.

[0127] At the DNA level, dual color-dual probe fluorescence in situhybridization (FISH) strategies are available to detect the BCR-ABL1translocation on metaphase chromosomes and interphase nuclei (Bentz etal., Blood, 83:1922-1928 (1994); Sinclair et al., Blood, 90:1395-1402(1997); Buno et al., Blood, 92:2315-2321 (1998)). With conventionalFISH, the initial strategy was to have the probe for the ABL1 oncogeneregion (distal of the breakpoint and ˜200kb in size) detected in onecolor (i.e., red) and the BCR region (proximal of the breakpoint) inanother color (i.e., green). Thus, in derivative chromosome 22 positivecells, one red and one green signal co-localized to give a yellowhybridization signal indicating the presence of a derivative 22chromosome while the normal chromosome 9 and chromosome 22 remained asindependent red and green signals. More recently, larger cloned DNAprobes that span both sides of the ABL1 translocation breakpoint havebeen utilized for detecting translocations. In this strategy, the partof the probe that is proximal to the breakpoint remains on the abnormalchromosome 9 and the part distal to the breakpoint co-localizes with BCRas in the previous strategy. This results in an extra signal (ES) on thetranslocated chromosome 9 and is the strategy behind the BCR-ABL1 ESdual-color translocation probe (Vysis, Inc., Downers Grove, Ill.) thatmany clinical cytogenetics laboratories use (Herens et al., Br. J.Haem., 110(1):214-216 (2000); Sinclair et al, Blood, 95(3):738-744(2000)). In the ES system, the ABL1 probe cocktail spans a genomictarget significantly larger than the ABL1 gene. This cocktail extendsfrom arginosuccinate synthetase gene (ASS), which is ˜250 kb upstreamfrom ABL1, through the ABL1 gene and several kb downstream.

[0128] Several single copy probes were designed for ABL1 and BCR byidentifying the boundaries of single copy intervals at these loci. FIGS.13 and 14 indicate the locations of potential single copy probes withinthe BCR and ABL1 loci, respectively. Eleven intervals exceeding 2 kblength are distributed throughout the BCR gene, 5 of which have beencurrently designed as probes. A similar number of additional shortersingle copy regions in this gene (between 1.5 and 2.0 kb) could becombined to more precisely delineate translocation breakpoints. In theABL1 gene, 10 intervals >2 kb are found, six of which have been designedas probes.

[0129] Multiple single copy probes for both BCR (FIG. 13) and ABL1 (FIG.14) have been deduced and oligonucleotide primer sets derived. For BCR,these probes discriminate between the minor (FIG. 13) and majorbreakpoints (FIG. 13 and SEQ ID Nos. 510-515). Conventional FISH testingwith BCR-ABL1 ES probe does not distinguish between rearrangements atthe major (M) and minor (m) breakpoints in the BCR gene. A mixture ofm-BCR probes detects the minor breakpoint often seen in patients withALL, and those designated M-BCR (SEQ ID Nos. 510-515) detect the majorbreakpoint evident in most patients with CML. If all of the probestranslocate to the chromosome 9, then the gene is interrupted at theminor breakpoint. If only the M-BCR probes translocate from chromosome22 to the derivative chromosome 9 and the m-BCR probes remain on thederivative chromosome 22, then the gene is interrupted at the majorbreakpoint.

[0130] For ABL1, two of the probes are predicted to be proximal of thebreakpoint (SEQ ID Nos. 516/517 and 518/519), and the others are distalto the breakpoint (SEQ ID Nos. 520 through 531). Hybridization of threeABL1 probes distal to the breakpoint in CML shows that the probes havemoved to the derivative chromosome 22 (FIG. 15), whereas a combinationof five ABL1 probes that span the breakage interval demonstrates thatsome probes remain on the derivative chromosome 9 while others move tothe derivative chromosome 22 (FIG. 16). Based on these results and thepositions of these probes, it can be deduced that the breakpointinterval spans positions 11004 bp through 65951 bp of FIG. 14. It willbe evident to one skilled in the art that the region of breakage can bemore precisely refined by hybridizing probes from the single copyintervals between Seq. ID Nos. 519 and 520. The exact location at thebreakpoint can be then determined from the genomic sequence of therefined breakage region.

[0131] Recent studies (Herens et al, Br. J Haem., 110:214-216 (2000))utilizing the commercially available ES probe have demonstrated that˜10% of CML patients do not have an extra hybridization signal on thederivative chromosome 9 because sequences are deleted upstream of theABL1 gene. In some instances, the deletions extend as far as the ASSgene and such a deletion is associated with poor prognosis (e.g., blastcrisis). These large FISH probes are not useful for detectinginterstitial deletions in the region between ASS and ABL1, as evidencedby an increased deletion detection rate of up to ⅓ of CML patients whenshorter probes of 100-200 kb are hybridized (Sinclair et al., Blood,95(3):738-744(2000)). Since some patients harbor deletions of sequencesproximal to the ABL1 breakpoint on the derivative or translocatedchromosome 9, single copy probes are being used to delineate the extentof hemizygosity in this chromosomal region. Correlation of deletionbreakpoints with clinical outcomes will determine if the loss ofspecific genes in this chromosonal interval is prognostic for clinicalfindings such as early blast crisis.

EXAMPLE 5

[0132] This example demonstrates that increased hybridization signalscan be generated using probe sequences from duplicated genomic domains.Several single copy probe sequences were designed, which were a part ofhighly similar duplicon or triplicon domains as previously described.

[0133] Several probes from chromosome 16p13.1 (SEQ ID Nos. 536-543),close to the inversion breakpoint in Acute Myelogenous Leukemia—Type M4,contain sequences that are a near perfect triplication of sequences inthis region. In the genome draft sequence, two of these domains aretandemly arranged, separating the probe sequences by 40 kb, and a thirdtelomeric interval is separated by 1.2 mb. The sequences of the threeintervals differ by only ˜1.5%. Hybridization with this probedemonstrated two clustered, but clearly separable signals. Onehybridization corresponds to the combined first and second paralogs andthe other to the third copy of this sequence.

[0134] A probe from the chromosome 17p11.2 interval that is commonlydeleted in Smith-Magenis Sydrome (SEQ ID Nos. 586-587) contained anear-perfect triplication. The probe was intended to detect a deletionwithin a near single-copy sequence in IVS4 of the SHMT1 gene. However,two paralogous subsequences separated by ˜15 kb, exhibiting 99.8%identity with the SMHT1 sequence, were also detected in the genome draftbetween the ZNF127 and PAIP1 genes ˜2.7 mb centromeric but also withinthe genetic interval commonly deleted in patients with this disorder.Due to the proximity of these sequences to the chromosome 17 centromere(which is a highly condensed region), a single hybridization locus wasobserved.

[0135] Two probes from the Down syndrome critical region (SEQ ID Nos.504/505-508/509) were each embedded in the same large duplicon, whichwas ≧80 kb in length. These duplicated sequences are separated by 1.1mb, and reside, respectively at the centromeric and telomeric ends ofchromosome band 21q22.2. Despite the fact that the duplicated sequenceswere separated by 1.1 mb, a single hybridization signal was detected inthis region of chromosome 21 using each probe. Therefore, this region ofchromosome 21, like chromosome 17p11.2, seems more condensed inmetaphase chromosomes than sequences in 16p13.1, in which duplicatedregions separated by similar distances were distinguishable.

[0136] Single copy probes developed from such regions, of course, lackknown repetitive sequence elements. However the probes generallyhybridize to all of the paralogous copies, since each of the copiesremain hybridized even under the most stringent hybridization washconditions. Because multiple, tightly clustered sites on the chromosomeare hybridized in a specific interval, the hybridization signal producedfrom these hybridizations is brighter than that expected from acomparable probe sequence which was represented once per haploid genome.Thus, these genomic duplicons or triplicons increase the effectivetarget size of the probe. This implies that shorter probes from suchregions can produce hybridization signals comparable in intensity tothose generated by longer probes. Selection of shorter probes fromduplicated genomic domains will be particularly useful for developmentof probes for genomic regions where long single copy intervals areunderrepresented.

EXAMPLE 6

[0137] The increasing availability of accurate draft human genomesequences has facilitated development of single copy probes inaccordance with the invention for many previously inaccessiblechromosomal regions. Although the most current comprehensive up-to-datesequence databases have been used to detect repetitive elements(http://wwvw.girinst.org/repbase) present in these draft sequences,hybridization of single copy probes to metaphase chromosomes hasrevealed that several probes contain previously unrecognized repetitivesequences. This was determined by documenting hybridization of a probeto the homologous chromosomal band where it is known to be mapped aswell as other locations not found in the draft genome sequence.

[0138] The draft genome sequence is incomplete, with ˜90% of theeuchromatic genome having been sequenced (International Human GenomeSequencing Consortium, Initial Sequencing and Analysis of the HumanGenome, Nature, 409:860-922, (2001). It was anticipated that somerepetitive sequence families, especially those present among the missingsequences, would have not been detected. Despite screening for knownrepetitive sequences, several euchromatic single copy probes appear tocontain homologs of repetitive sequence families that are predominantlyfound in multiple copies on the short arms of acrocentric chromosomes(chromosomes 13, 14, 15, 21, and 22). These probes included threesequences derived from the Down Syndrome critical region on chromosome21 (amplified with SEQ ID Nos. 504/505, 506/507 and 508/509), twosequences from chromosome 16p13.1 that straddle the site of chromosomalinversion in Acute Myelogenous Leukemia, Type M4 (amplified with SEQ IDNos. 536/537 and 538/539).

[0139] Such chromosomal domains, termed nucleolar organizer regions, areknown to contain thousands of copies of the ribosomal RNA cistronsarranged in long tandem arrays (Sylvester et al., Hum. Genet.,; 73:193-8(1986)). The human genome draft genome sequence is devoid of contiguoussequences from these chromosomal regions. The International SequencingConsortium eliminated clones containing these and other tandemlyrepeated sequences (e.g., heterochromatin) from consideration early inthe sequencing effort, since it was recognized that any assembly ofsequences from such clones would be ambiguous, and thus unreliable.Other sequences distinct from, but co-localizing with, ribosomal RNAgenes would most likely also be tandemly arranged in chromosomalnucleolar organizer regions. Because of the lack of sequence informationfrom these intervals, the distribution of sequences within them that arerelated to sequences elsewhere in the genome has not been previouslyappreciated. While single copy FISH with these probes demonstratedlocalization to the expected euchromatic intervals, additionalsignificant hybridization to the short arms of several acrocentricchromosomes was seen. Addition of C₀t 1 DNA prior to hybridizationremoved cross-hybridization to these repetitive sequences. Theseadditional signals are consistent with tandemly organized multiplecopies of sequences related to the probe on short arms of acrocentricchromosomes.

[0140] In addition, hybridization of presumed single copy probes tointerspersed repetitive sequence families was detected, despite the factthat these probes were filtered for repetitive sequences usingRepeatMasker software. One probe, mapping to chromosome 17p11.2, withinthe interval commonly deleted in Smith-Magenis syndrome (amplified withSEQ ID No. 586/587), was found to cross-hybridize with interspersedrepeats. Sequences close to the translocation breakpoint at chromosome9q34 (amplified with SEQ ID Nos. 518/519 and 526/527) also potentiallycontain interspersed repetitive sequences, based on hybridization ofcombinations of these probes with another probe from this region. Thehybridization signals at the mapped locations for these probes were notstronger than those observed for the cross-hybridizing sequences, norwere the cross-hybridizing sequences removed by increasing thestringency during the washing procedures. This suggests that the probescontain previously unrecognized repetitive sequence families, ratherthan highly divergent copies of known interspersed repeats (whosefailure to be recognized by RepeatMasker software would have led totheir inadvertent inclusion in the designed probe).

[0141] Although all of these probes appear to contain members ofpreviously unrecognized repetitive sequences families, the probesthemselves are likely to be composed of both single copy and repetitivesequences. It is feasible to separate these sequence components byiterative hybridization of different PCR-generated subsets of each probesequence to chromosomal DNA. However, since each entire probe sequenceis known, the sequence can be added to the repeat sequence database usedto develop new, additional single copy probes. The probe sequences arenot lengthy (some interspersed repeat families are, in fact, longer,e.g., L1, than the longest single copy probe); therefore, minimaladditional computational overhead is incurred by addition of thesesequences to the database of human repetitive sequence families. Theaddition of these previously unknown repetitive sequence family membersresults in a more comprehensive repetitive sequence database, that inturn improves the design of single copy probes. Single copy probessubsequently designed using the larger repeat sequence database, willnot contain these novel repetitive sequences. This heuristic algorithmimproves the purity of single copy sequences in single copy probes.

[0142] Generally speaking, the invention thus provides a method ofdetermining the existence of previously unknown repeat sequence familiesin a genome. This method involves reacting a labeled, putative singlecopy nucleic acid probe with the genome, and causing the probe tohybridize. If the probe hybridizes at more than three differentlocations (and preferably at more than ten different locations), then itis likely that a new, previously unknown repeat sequence has been found.

[0143] All references cited above are expressly incorporated byreference herein. In addition, the subject matter of Disclosure Document#471449 filed Mar. 27, 2000, is also incorporated by reference herein.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030224356). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

We claim:
 1. A nucleic acid hybridization probe comprising a labeled,single copy nucleic acid which will hybridize to a deduced single copysequence interval in target nucleic acid of known sequence, said nucleicacid probe having a length of at least about 50 nucleotides.
 2. Theprobe of claim 1, said probe including a plurality of different, labelednucleic acids each of which will hybridize to respective deduced singlecopy sequence intervals in said target nucleic acid, each of saidnucleic acid probes having a length of at least about 50 nucleotides. 3.The probe of claim 1, said nucleic acid probe having a length of atleast 100 nucleotides.
 4. The probe of claim 3, said nucleic acid probehaving a length of at least about 2000 nucleotides.
 5. The probe ofclaim 1, said target nucleic acid being selected from the groupconsisting of DNA, RNA and mRNA.
 6. The probe of claim 5, said targetnucleic acid being DNA.
 7. The probe of claim 1, said nucleic acid probebeing single stranded.
 8. The probe of claim 1, said probe beingessentially free of blocking nucleic acid sequences which will hybridizeto repeat sequences within the genome of which said target nucleic acidis a part.
 9. The probe of claim 1, said nucleic acid probe beinglabeled with a label selected from the group consisting offluorochrome-responsive labels, fluorochromes, colorimetric chemical,conjugated proteins, antibodies, antigens, and mixtures thereof.
 10. Theprobe of claim 9, said nucleic acid probe being labeled with afluorochrome-responsive label.
 11. The probe of claim 1, there being atleast about 80% sequence identity between said probe and a sequencewhich is a complement to said target sequence.
 12. The probe of claim11, said probe being complementary to said target sequence.
 13. In ahybridization method including the steps of preparing a reaction mixturecomprising a target nucleic acid sequence and a nucleic acid probe whichhybridizes to at least a portion of said target nucleic acid sequence,and causing said probe to hybridize to said target nucleic acidsequence, the improvement which comprises using as said probe a labeled,single copy nucleic acid which hybridizes to a deduced single copysequence interval in target nucleic acid of known sequence, said nucleicacid probe having a length of at least about 50 nucleotides.
 14. Themethod of claim 13, said probe including a plurality of different,labeled nucleic acids each of which hybridizes to respective deducedsingle copy sequence intervals in said target nucleic acid, each of saidnucleic acid probes having a length of at least about 50 nucleotides.15. The method of claim 13, said nucleic acid probe having a length ofat least 100 nucleotides.
 16. The method of claim 15, said nucleic acidprobe having a length of at least about 2000 nucleotides.
 17. The methodof claim 13, said target nucleic acid being selected from the groupconsisting of DNA, RNA and mRNA.
 18. The method of claim 17, said targetnucleic acid being DNA.
 19. The method of claim 13, said nucleic acidprobe being single stranded.
 20. The method of claim 13, said probebeing essentially free of blocking nucleic acid sequences whichhybridizes repeat sequences within the genome of which said targetnucleic acid is a part.
 21. The method of claim 13, said nucleic acidprobe being labeled with a label selected from the group consisting offluorochrome-responsive labels, fluorochromes, calorimetric chemical,conjugated proteins, antibodies, antigens, and mixtures thereof.
 22. Themethod of claim 21, said nucleic acid probe being labeled with afluorochrome-responsive label.
 23. The method of claim 13, saidhybridization method selected from the group consisting of in situhybridization, Southern blot, and other methods in which nucleic acid isimmobilized.
 24. The method of claim 13, there being at least about 80%sequence identity between said probe and a sequence which is acomplement to said target sequence.
 25. The method of claim 24, saidprobe being complementary to said target sequence.
 26. A method ofdeveloping a hybridization probe for a target nucleic acid sequenceforming a part of a genome, said method comprising the steps of:determining the sequence of at least one single copy sequence in saidtarget nucleic acid sequence; and developing a hybridization probe whichhybridizes to at least a part of said single copy sequence.
 27. Themethod of claim 26, including the steps of: determining the sequence ofsaid target nucleic acid sequence; determining the repeat sequencesfound in said genome; and comparing said sequence of said target nucleicacid sequence and said repeat sequences in order to determine saidsequence of said at least one single copy sequence.
 28. The method ofclaim 26, said probe developing step comprising the steps of obtainingat least a part of said single copy sequence, and purifying said part ofsaid single copy sequence.
 29. The method of claim 28, said purifyingstep comprising carrying out PCR.
 30. The method of claim 26, includingthe step of labeling said hybridization probe.
 31. The method of claim26, target nucleic acid sequence and said probe being DNA.
 32. Themethod of claim 26, said hybridization probe having at least about 80%sequence identity with said single copy sequence.
 33. The method ofclaim 32, said hybridization probe being complementary to single copysequence.
 34. A DNA sequence having at least about 80% sequence identitywith a member selected from the group consisting of SEQ ID Nos. 429-446.35. The DNA sequence of claim 34, said sequence having at least about90% sequence identity.
 36. A DNA sequence selected from the groupconsisting of SEQ ID Nos. 429-446 and 480-613.
 37. A DNA sequence havingat least about 80% sequence identity with a PCR-amplified sequenceproduced using as PCR primers adjacent pairs of the sequences identifiedas SEQ ID Nos. 429-446 and 480-613, beginning with SEQ ID No.
 429. 38.The sequence of claim 37, there being at least about 90% sequenceidentity.
 39. A method of determining the existence of previouslyunknown repeat sequence families in a genome, comprising the steps ofreacting a labeled, putative single copy nucleic acid probe with thegenome, causing the probe to hybridize, and ascertaining if the probehybridizes to the genome at more than three different locations as adetermination of a new repeat sequence family.
 40. The method of claim39, including the step of ascertaining if the probe hybridizes at morethan ten different locations.
 41. The probe of claim 1, said nucleicacid derived from a duplicon or triplicon sequence interval.
 42. Themethod of claim 13, including the step of selecting a single copynucleic acid which will hybridize to a duplicon or triplicon sequencedomain.
 43. The method of claim 26, said determining step comprising thestep of selecting said single copy sequence from a duplicon or tripliconsequence domain.
 44. A method of determining a chromosome breakpoint,comprising the steps of: providing a pair of separate, labeled, singlecopy nucleic acid probes of predetermined sequence and designed torespectively hybridize on opposite sides of said breakpoint; reactingsaid pair of probes with a chromosomal target sequence containing saidbreakpoint, and causing the probes to hybridize to the target sequence;and detecting said hybridized probes as a way of ascertaining thelocation of said breakpoint.
 45. The method of claim 44, including thestep of providing a plurality of separate, labeled, single copy nucleicacid probes of predetermined sequence designed to hybridize on one sideof said breakpoint, and providing a plurality of separate, labeled,single copy nucleic acid probes of predetermined sequence designed tohybridize on the other side of said breakpoint.